Duplex stabilizing fluorescence quenchers for nucleic acid probes

ABSTRACT

Diaryl-azo derivatives are efficient fluorescence quenchers as well as nucleic acid duplex-stabilizing agents and are useful in oligonucleotide conjugates and probes. The oligonucleotide-quencher conjugates may be used in detection methods for nucleic acid targets.

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/546,965, filed Aug. 17, 2017, entitled “Duplex StabilizingFluorescence Quenchers for Nucleic Acid Probes,” the entire contents ofwhich are hereby incorporated by reference.

BACKGROUND

This disclosure pertains to oligonucleotide-quencher conjugates withimproved fluorescence characteristics, and to reagents suitable forincorporating novel quencher moieties into oligonucleotides. Thedisclosure also pertains to the use of oligonucleotide-quencherconjugates in detection methods for nucleic acid targets.

Minor groove binding (MGB) and fluorescence quenching are two keyfeatures of fluorescence-based DNA probe technologies. The first featureprovides improved duplex stabilization and hybridization specificity,whereas the second one is required for reduction of backgroundfluorescence of unhybridized probes. Historically, two independentfunctional groups have been used to fulfill these functions. The MGB andquenchers have been introduced into DNA probes using two reagents suchas MGB-modified DNA synthesis solid supports and Quencherphosphoramidites. In another approach a MGB-Quencher solid support isutilized wherein MGB and quencher are linked together via a flexiblespacer with each group serving its unique function. The existingMGB-Quencher structures and respective DNA synthesis reagents asdisclosed in U.S. Pat. No. 6,492,346 are complicated and costly tomanufacture.

SUMMARY

The present disclosure relates to certain diaryl-azo derivatives whereinsaid derivatives are efficient fluorescence quenchers as well as nucleicacid duplex-stabilizing agents. The present disclosure also relates tooligonucleotide conjugates bearing said diaryl-azo derivatives.

It is considered desirable to combine Minor groove binding (MGB) andfluorescence quenching in a simple unified structure. One approach tothis problem is to convert part of the MGB moiety into a fluorescencequencher as Illustrated in FIG. 1. It is, however, not clear that suchfunctional unification can be achieved within one structure withoutaffecting the minor groove binding or fluorescence quenching properties.Surprisingly, certain preferred embodiments described herein cansuccessfully accomplish this goal.

The disclosed diaryl-azo derivatives are especially useful formodification of certain types of known MGB agents (such as oligomers of3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid(CDPI)) wherein a portion of the MGB structure is replaced with adisclosed diaryl-azo derivative. The two portions of such hybridmolecules are connected via a rigid amide bond with no ability forinternal folding and limited rotation and, therefore, act as a singleentity. As demonstrated herein, certain types of such hybrid moleculesare as efficient or more efficient duplex stabilizing agent than theirparent MGB moieties. The resultant hybrids are also efficientfluorescence quenchers for variety of typical fluorophores. This abilityto merge duplex stabilization and fluorescence quenching within onestructure is the key point of the disclosure.

The availability of the disclosed derivatives eliminates the need fortwo separate reagents and simplifies preparation of oligonucleotideconjugates that require both enhanced hybridization and efficientfluorescence quenching. Such conjugates are of special interest influorescence-based diagnostic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the concept of merging a minor groove binderand fluorescence quencher into a single Duplex-Stabilizing Quencher(DSQ) moiety.

FIG. 2 shows a generic synthetic Scheme 1 for the preparation ofpentafluorophenyl (PFP) esters g9 and synthesis supports g10.

FIG. 3A shows an analytical HPLC of the T8-477 oligonucleotide conjugateprepared according to generic Scheme 1.

FIG. 3B shows photo diode array (PDA) UV-VIS spectra of the main peak ofthe T8-477 oligonucleotide conjugate prepared according to genericScheme 1.

FIG. 4A shows structures and absorption maxima of the T8 oligonucleotideconjugates synthesized according to reaction Scheme 1.

FIG. 4B shows structures and absorption maxima of the T8 oligonucleotideconjugates synthesized according to reaction Scheme 1.

FIG. 4C shows structures and absorption maxima of the T8 oligonucleotideconjugates synthesized according to reaction Scheme 1.

FIG. 4D shows structures and absorption maxima of the T8 oligonucleotideconjugates synthesized according to reaction Scheme 1.

FIG. 5A shows steps in reaction Scheme 2, synthesis of polystyrenesupport ID #478.

FIG. 5B shows steps in reaction Scheme 2, synthesis of polystyrenesupport ID #478.

FIG. 6A shows an analytical HPLC of the T8-oligonucleotide conjugatesynthesized starting from the polystyrene support ID #478.

FIG. 6B shows PDA UV-VIS spectra of the main peak and product structureof the T8-oligonucleotide conjugate synthesized starting from thepolystyrene support ID #478.

FIG. 7A shows steps in reaction Scheme 3, synthesis of polystyrenesupport ID #480.

FIG. 7B shows steps in reaction Scheme 3, synthesis of polystyrenesupport ID #480.

FIG. 8A shows an analytical HPLC of the T8-oligonucleotide conjugatesynthesized starting from the polystyrene support ID #480.

FIG. 8B shows PDA UV-VIS spectra of the main peak and product structureof the T8-oligonucleotide conjugate synthesized starting from thepolystyrene support ID #480.

FIG. 9A shows steps in reaction Scheme 4, synthesis of phosphoramidite75.

FIG. 9B shows steps in reaction Scheme 4, synthesis of phosphoramidite75.

FIG. 10A shows analytical HPLC of the T8-oligonucleotide conjugatesynthesized using phosphoramidite 75.

FIG. 10B shows PDA UV-VIS spectra of the main peak and product structureof the T8-oligonucleotide conjugate synthesized using phosphoramidite75.

FIG. 11A shows UV-VIS absorption spectra of T8-DSQ conjugates.

FIG. 11B shows UV-VIS absorption spectra of T8-DSQ conjugates.

FIG. 11C shows UV-VIS absorption spectra of T8-DSQ conjugates.

FIG. 12 shows melting temperatures of (KPC+KPC-C) and (IC+IC-C) duplexesand corresponding sequences.

FIG. 13 shows background fluorescence (90° C.) of KPC and IColigonucleotide probes labeled with representative 3′-DSQ derivatives,where the probes were also labeled with 5′-FAM (KPC) or 5′-AP525 (IC).

FIG. 14A shows the effects of various DSQ ligands on the d(T₈)/d(A₈)duplex stability.

FIG. 14B shows the effects of various DSQ ligands on the d(T₈)/d(A₈)duplex melting temperature.

FIG. 15 shows a comparison of duplex-stabilizing effects of CDPI₂, CDPI₃(ID #391), MGB-Quencher (ID #385) and DSQ (ID #477) in differentsequence context, and corresponding sequences.

FIG. 16 shows PCR performance of two TaqMan probes labeled withrepresentative DSQ derivatives.

FIG. 17A shows duplex and single strand fluorescence temperature profileof an oligonucleotide conjugate labeled with 5′-DSQ ID #473 and 5′-FAM.

FIG. 17B shows duplex and single strand fluorescence temperature profileof an oligonucleotide conjugate labeled with 5′-DSQ ID #473 and5′-AP525.

FIG. 18 shows the structures of MGB-Quencher (ID #385), CDPI₃ (ID #391),AP525, AP593 and AP639 dyes, with a wavy bond indicating the attachmentpoint to an oligonucleotide.

DETAILED DESCRIPTION Definitions

The term “diaryl-azo” compounds or derivatives refers to diazene (HN═NH)derivatives wherein both hydrogen atoms substituted with two arylgroups. For the purpose of this specification the term aryl encompassesthe definitions of both terms “aryl” and “heteroaryl”.

The term “aromatic amino acid” refers to an aromatic compound comprisingan aromatic carboxylic and an aromatic amino group.

The abbreviations MGB, Fl, Q, CPG and ODN refer to “minor groovebinder”, “fluorescent label” or “fluorophore”, “quencher”, “controlledpore glass” (as an example of a solid support) and “oligonucleotide”moieties or molecules, respectively, and in a manner which is apparentfrom context.

The term “minor groove binder” refers to a moiety that is capable offorming a complex (typically non-covalent) with the minor groove of DNA.The minor groove binders of the invention are oligonucleotide conjugates(or “probes”) as described in U.S. Pat. No. 5,801,155 and U.S. Pat. No.6,312,894, both hereby incorporated by reference. These conjugates formhyper-stabilized duplexes with complementary DNA. In particular,sequence specificity of short minor groove binder probes is excellentfor high temperature applications such as PCR. The probes/conjugates ofthe present disclosure can also have a covalently attached minor groovebinder. A variety of suitable minor groove binders have been describedin the literature (U.S. Pat. No. 5,801,155; Wemmer, D. E., and Dervan P.B. (1997; Walker et al. (1997), Biopolymers, 44:323-334 (1997); Zimmer,C & Wahnert, U., (1986) and Reddy, et al. (1999), Pharmacol. Therap.,84:1-111 (1999)).

Suitable methods for attaching minor groove binders (as well as reportergroups such as fluorophores and quenchers) through linkers tooligonucleotides have also been described (U.S. Pat. Nos. RE 38,416;5,512,667; 5,419,966; 5,696,251; 5,585,481; 5,942,610 and 5,736,626).

The term “fluorescent label or fluorophore” refers to an organic moietythat is capable of absorbing and re-emitting light. Typically,fluorophores absorb light of certain wavelength range (excitationspectrum) and re-emitting it at a longer wavelength range (emissionspectrum) with respective excitation and emission maxima. Thefluorophores of the invention have excitation and emission maximabetween 400 and 900 nm. Examples of these dye classes can be found inHaugland, et al., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS,SIXTH ED., Molecular Probes, Eugene, Oreg. 1996; Krasovitskii andBolotin, ORGANIC LUMINESCENT MATERIALS, VCH Publishers, N.Y., 1988;Zolliger, COLOR CHEMISTRY, 2nd Edition, VCH Publishers, N.Y., 1991.Still other dyes are provided via online sites such as zeiss.com.Phosphonate dyes are disclosed in co-owned U.S. Pat. No. 7,671,218, U.S.Pat. No. 7,767,834 and U.S. Pat. No. 8,163,910B2

The term “quencher” refers to an organic moiety that is capable ofreducing the efficiency of light re-mission by a fluorophore. Quenchershave been disclosed in U.S. Pat. No. 3,996,345, in co-owned U.S. Pat.Nos. 6,727,356 and 6,790,945, and in Matayoshi et al., 1990.

The term “oligonucleotide” refers to a fragment of natural or artificialnucleic acid or combination of thereof. Examples of artificial nucleicacids include analogs with modified sugar-phosphate backbone such as2-OMe nucleic acid, peptide nucleic acid (PNA), locked nucleic acid(LNA), threose nucleic acid (TNA), glycol nucleic acid (GNA) (U.S. Pat.No. 5,539,082, U.S. Pat. Nos. 8,293,684 and 9,464,316). Artificialnucleic acid (U.S. Pat. No. 9,169,256) may also comprise modifiednucleobases.

The term “modified nucleobases or modified bases” refers to those basesthat differ from the naturally-occurring bases (adenine, cytosine,guanine, thymine, and uracil) by addition or deletion of one or morefunctional groups, differences in the heterocyclic ring structure (i.e.,substitution of carbon for a heteroatom, or vice versa), and/orattachment of one or more linker arm structures to the base. Modifiedbases include naturally-occurring and synthetic modifications andanalogues of the major bases such as, for example, hypoxanthine,2-aminoadenine, 2-thiouracil, 2-thiothymine, inosine,5-N⁴-ethenocytosine, 4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine. Any modified nucleotide ornucleotide analogue compatible with hybridization of probe with anucleic acid conjugate to a target sequence is useful, even if themodified nucleotide or nucleotide analogue itself does not participatein base-pairing, or has altered base-pairing properties compared tonaturally-occurring nucleotides. Examples of modified bases aredisclosed in U.S. Pat. Nos. 7,045,610; 5,824,796; 6,127,121; 5,912,340;and PCT Publications WO 01/38584 and WO 01/64958, each of which ishereby incorporated herein by reference in its entirety. Preferredmodified bases include 5-hydroxybutynyl uridine for uridine;4-(4,6-Diamino-¹H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol,4-amino-¹H-pyrazolo[3,4-d]pyrimidine, and4-amino-¹H-pyrazolo[3,4-d]pyrimidine for adenine;5-(4-Hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione for thymine; and6-amino-¹H-pyrazolo[3,4-d]pyrimidin-4(5H)-one for guanine. Particularlypreferred modified bases are “Super A®:4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol,” “SuperG®: 4-hydroxy-6-amino pyrazolopyrimidine” (elitechgroup.com) and “SuperT®: 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione”. “Super-D™:3-Alkynyl pyrazolopyrimidine” analogues as universal bases are disclosedin U.S. Patent Application Publication No. 2012/0244535, incorporated byreference.

The term “linker” and “linking group” refers to a moiety that is used toassemble various portions of the molecule or to covalently attach themolecule (or portions thereof) to a solid support. Typically, a linkeror linking group has functional groups that are used to interact withand form covalent bonds with functional groups in the ligands orcomponents (e.g., fluorophores, oligonucleotides, minor groove binders,or quenchers) of the oligonucleotide probes described and used herein.Additionally, a linker can include linear or acyclic portions, cyclicportions, aromatic rings or combinations thereof (U.S. Pat. Nos.5,419,966; 5,696,251; 5,585,481; 5,942,610 and 5,736,626).

The terms “functional” and “reactive” groups in this invention are usedinterchangeably and refer to chemical groups and moieties that aresuitable for the formation of a chemical bond. They are exemplified butnot limited to amines, oxyamines, hydrazines, hydrazides,semi-carbazides, semi-thiocarbazides, hydroxyl-substituted compounds,sulfur compounds (such as thiols, dithiols, thiocarbonyl compounds,phosphorothiates), carboxylates, phosphates, phosphonates, aromaticnitrogens (such as in pyridine), amide nitrogens, azides, electron-richaromatics, etc.), acids (in the presence of activating agents), esters,imidoesters, anhydrides, acid chlorides, acyl azides, lactones,azlactones, isocyanates, isothiocyanates, o-acylisoureas, acid amides(such as acyl imidazolides or phosphoramidites), carbonyl compounds,halogenated hydrocarbons, halogenated aromatics (such as triazinechloride, electron-deficient fluoroaromatics), unsaturated hydrocarbons,aromatic diazonium salts, epoxides, aziridines. Other types offunctional or reactive groups include photo-reactive (azides,benzophenones, diazirines, etc.), metal chelating groups (aminodiaceticacid), substrates for metal-catalyzed coupling, ligands for molecularrecognition (such as biotin), antigens and haptens. Functional andreactive groups of this invention may also be used in conjunction withbi-functional or poly-functional cross-linking reagents (such asbis-amines, bis-aldehydes, maleimido-NHS esters, etc). Other examples ofreactive groups and cross-linking reaction can be found in literature(Hermanson, Bioconjugate Techniques, Elsevier, 1996).

“Leaving group” means a molecular fragment that departs with a pair ofelectrons in heterolytic bond cleavage. Common leaving groups arehalides such as Cl—, Br—, and I—, sulfonate esters such as tosylate(TsO—), phenolates such as 4-nitrophenylate, and water (Smith, 2007).

“Protecting group” or “protected form thereof” or “protected functionalgroup” or “PFP” or “blocking group” refers to a grouping of atoms that,when attached to a reactive group in a molecule, masks, reduces orprevents that reactivity. Examples of protecting groups can be found inT. W. Greene and P. G. Wuts, 2007 and Harrison and Harrison et al 1971to 1996. Representative amino protecting groups include formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilylethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC)and the like. Representative hydroxy protecting groups include thosewhere the hydroxy group is either acylated or alkylated such as benzyland trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers,trialkylsilyl ethers and allyl ethers.

The term “solid support” or “synthesis solid support” refers to anysupport that is compatible with oligonucleotide synthesis including, forexample, glass, controlled pore glass, polymeric materials, polystyrene,beads, coated glass and the like.

The term “exocyclic amino group” means an amino group situated outsideof a chemical ring, such as in aniline.

The term “alkyl” refers to a linear, branched, or cyclic saturatedmonovalent hydrocarbon substituent or a combination of cyclic and linearor branched saturated monovalent substituents having the number ofcarbon atoms indexed in the prefix. For example, (C₁-C₈)alkyl is meantto include methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl,cyclopentyl, cyclopropylmethyl and the like. For each of the definitionsherein (e.g., alkyl, alkenyl, alkoxy, aralkyloxy), when a prefix is notincluded to indicate the number of chain carbon atoms in an alkylportions, the substituent thereof will have eight or fewer main chaincarbon atoms.

The term “alkylene” refers to a linear saturated divalent hydrocarbonsubstituent or a branched saturated divalent hydrocarbon substituenthaving the number of carbon atoms indicated in the prefix. For example,(C₁-C₆)alkylene is meant to include methylene, ethylene, propylene,2-methylpropylene, pentylene, and the like.

The term “aromatic” or “aryl” means a monovalent or bivalent (e.g.,arylene) monocyclic, bicyclic aromatic or tricyclic hydrocarbonsubstituent of 5 to 14 ring atoms which is unsubstituted or substituted.If substituted the substituents are selected from those groups providedbelow. The term “heteromatic” or “heteroaryl” refers to aryl wherein oneor more heteroatoms or heteroatom functional groups have replaced a ringcarbon, while retaining aromatic properties, e.g., pyridyl, quinolinyl,quinazolinyl, thienyl, and the like. More specifically the terms aryland heteroaryl include, but are not limited to, phenyl, 1-naphthyl,2-naphthyl, thienyl, thiazolyl and benzothiazolyl, and the substitutedforms thereof.

Substituents for the aryl and heteroaryl groups are varied and areselected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂,—CO₂R′, —CONR′R″, —C(O)R′, —OC(O) NR′R″, —NR″C(O)R′, —NR″C(O)2R′,—NR′—C(O), NR″R′″, —NH—C(NH2)=NH, —NR′C(NH2)=NH, —NH—C(NH2)=NR′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy,and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″and R′″ are independently selected from hydrogen, (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. Preferredsubstituents are —OH, Halogen, OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN and—NO₂— where R′ and R″ are independently —H— or —(C₁-C₄).

The prefix “halo” and the term “halogen,” when used to describe asubstituent, refer to —F, —Cl, —Br and —I. Certain compounds oroligonucleotides of the present disclosure may exist in a salt form.

Such salts include base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When the compounds or modified oligonucleotides of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromorganic acids like acetic, propionic, isobutyric, maleic, malonic,lactic, benzoic, succinic, suberic, fumaric, mandelic, phthalic,benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, andthe like. Also included are salts of amino acids such as arginate andthe like, and salts of organic acids like glucuronic or galactunoricacids and the like (Berge, S. M., et al. 1977). Certain specificcompounds described herein contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts. The neutral forms of the compounds may be regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentdisclosure.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and are intendedto be encompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present disclosure. The methods forthe determination of stereochemistry and the separation of isomers arewell-known in the art (March J. 1992).

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present disclosure, whether radioactive or not(e.g, ²H), are intended to be encompassed within the scope of thepresent disclosure.

“Optional” or “optionally” in the above definitions means that thesubsequently described event or circumstance may but need not occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “aryl optionallymono- or di-substituted with an alkyl group” means that the alkyl groupmay, but need not, be present, and the description includes situationswhere the aryl group is mono- or bis-substituted with an alkyl group andsituations where the aryl group is not substituted with the alkyl group.

In certain instances, amplification is carried out using a polymerase.The polymerase can, but need not, have 5′ nuclease activity. In certainother instances, primer extension is carried out using a reversetranscriptase and amplification is carried out using a polymerase (U.S.Pat. Nos. 6,312,894, and 7,381,818). In other instances, theamplification is isothermal (U.S. Patent Application No. 2014 0255928).

In one embodiment, the amplified targets are detected, with dual FRETDSQ-conjugute probes, as taught in U.S. Pat. Nos. 6,312,894, 7,381,818and U.S. Patent Application No. 2014 0255928, all incorporated byreference)

The term “digital PCR” refers to an approach to nucleic acid detectionand quantification, which is a method of absolute quantification sinceit directly counts the number of target molecules rather than relying onreference standards or endogenous controls (Sedlak and Jerome 2013).

The term “arrays” refers to hybridization of the probes of the inventionto an immobilized oligonucleotide (U.S. Pat. No. 6,045,996). In somearrays, the probes described herein are immobilized to a solid support(U.S. Pat. No. 6,821,727).

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques in organic chemistry, biochemistry,oligonucleotide synthesis and modification, bioconjugate chemistry,nucleic acid hybridization, molecular biology, microbiology, genetics,recombinant DNA, and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Maniatis, Fritsch & Sambrook 1982; Sambrook, Fritsch &Maniatis, (1989); Ausubel, et al., 1987, 1988, 1989, 1990, 1991, 1992,1993, 1994, 1995, 1996; Gait (ed.), 1984; Eckstein (ed.), 1991.

Description

Certain diaryl-azo derivatives are described herein, wherein saidderivatives are efficient fluorescence quenchers as well as nucleic acidduplex-stabilizing agents. These derivates may also be referred to asDuplex-Stabilizing Quenching (DSQ) compounds, derivatives or agents.

In preferred embodiments, DSQ derivatives comprise a diaryl-azocarboxylic acid of general Formula I:

wherein Ar¹ and Ar² are aromatic moieties, optionally comprising afunctional group or a linker with a functional group or protectedfunctional group.

In certain preferred embodiments, the diaryl-azo carboxylic acids ofFormula I are coupled via a carboxamide bond to a minor groove bindingaromatic amino acid (or peptide) of Formula II to yield DSQ compounds ofFormula III:

wherein Ar¹ and Ar² are aromatic or hetero-aromatic moieties, which areoptionally substituted with a functional group or a linker with afunctional group or protected functional group or a linker connecting toa synthesis solid support;Ar³ is an aromatic or hetero-aromatic moiety;R¹ is H, alkyl or alkyl covalently connected to Ar³;X is hydroxyl, a leaving group, a linker with a functional group orprotected functional group, or a linker connecting to a synthesis solidsupport; andn is from 0 to 5. If n is greater than 1 each Ar³ may be the same ordifferent.

Table 1 below shows examples of HOOC—Ar¹ and Ar² groups in accordancewith preferred embodiments disclosed herein, where the HOOC—Ar¹ and Ar²groups are substituted with preferred substituents. The wavy bondindicates the attachment point to the azo group in Formula III.

TABLE 1 HOOC—Ar¹

R¹, R², R³, R⁴ = —H, -Alkyl, -OAlkyl, -Aryl, -OAryl, —F, —Cl, —Br, —CF₃,—NO₂, R⁵ = H, Alkyl, —C(═O)OAlkyl, protecting group

R¹, R², R³ = —H, -Alkyl, -OAlkyl, -Aryl, -OAryl, —F, —Cl, —Br, —CF₃,—NO₂ X = O, S

R¹, R², R³, R⁴ = —H, -Alkyl, -OAlkyl, -Aryl, -OAryl, —F, —Cl, —Br, —CF₃,—NO₂,

R¹, R², R³ = —H, -Alkyl, -OAlkyl, -Aryl, -OAryl, —F, —Cl, —Br, —CF₃,—NO₂,

R¹, R² = —H, -Alkyl, -OAlkyl, -Aryl, -Heteroaryl, -OAryl, —F, —Cl, —Br,—CF₃, —NO₂, R⁵ = H, Alkyl

R¹ = —H, -Alkyl, -OAlkyl, -Aryl, -Heteroaryl, -OAryl, —F, —Cl, —Br,—CF₃, —NO₂, R⁵ = H, Alkyl

R¹ = —H, -Alkyl, -OAlkyl, -Aryl, -Heteroaryl, -OAryl, —F, —Cl, —Br,—CF₃, —NO₂, Ar²

R¹, R² = —H, -Alkyl, -Aryl, -Heteroaryl, Alkyl-OH, Alkyl-COOH,Alkyl-Amine R³, R⁴, R⁵, R⁶ = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl,-OAryl, —NHR¹, —NHC(═O)R¹,

R^(1,) R² = —H, -Alkyl, -Aryl, -Heteroaryl, Alkyl-OH, Alkyl-COOH,Alkyl-Amine R³, R⁴, R⁵, R⁶, R⁷, R⁸ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹

X = S, Se, O, NH, NCH₃ R¹, R² = —H, -Alkyl, -Aryl, -Heteroaryl,Alkyl-OH, Alkyl-COOH, Alkyl-Amine R³ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹

X = S, Se, O, NH, NCH₃ R¹, R² = —H, -Alkyl, -Aryl, -Heteroaryl,Alkyl-OH, Alkyl-COOH, Alkyl-Amine

X = S, Se, O, NH, NCH₃ R¹, R² = —H, -Alkyl, -Aryl, -Heteroaryl,Alkyl-OH, Alkyl-COOH, Alkyl-Amine R³, R⁴ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹

X = O, S, Se R¹, R³, R⁴, R⁵, R⁶ = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl,-OAryl, —NHR¹, —NHC(═O)R¹, Alkyl-OH, Alkyl-COOH, Alkyl-Amine

X = O, S, Se R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, Alkyl-OH, Alkyl-COOH, Alkyl-Amine

n = 2-4, k = 2-4

n = 2-4, R = H, Alkyl, Alkyl-OH, Alkyl-COOH, Alkyl-Amine

Table 2 below shows examples of minor groove binding aromatic aminoacids (HOOC—Ar³—NH(R¹)) of Formula II in accordance with preferredembodiments disclosed herein, where the minor groove binding aromaticamino acids are substituted with preferred substituents. The wavy bondindicates the attachment point to the azo group in Formula II.

TABLE 2 HOOC—Ar³—NH(R¹)

X = NH, N(Alkyl), N(C═O)OAlkyl, N(Protecting group), O, S, Se R¹, R², R³= H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, —F,—Cl, —Br

X = NH, N(Alkyl), N(C═O)OAlkyl, N(Protecting group), O, S, Se R¹, R²,R³, R⁴ = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl, -OAryl, —NHR¹,—NHC(═O)R¹, —F, —Cl, —Br

X = NH, N(Alkyl), O, S, Se R², R³ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, —F, —Cl, —Br

X = NH, N(Alkyl), O, S, Se R², R³ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, —F, —Cl, —Br R⁵ = —H, Alkyl

X = NH, N(Alkyl), O, S, Se R¹, R², R³, R⁴ = H, -Alkyl, -Aryl,Heteroaryl, -OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, —F, —Cl, —Br R⁵ = —H,Alkyl

X = NH, N(Alkyl), O, S, Se R¹, R³, R⁴ = H, -Alkyl, -Aryl, Heteroaryl,-OAlkyl, -OAryl, —NHR¹, —NHC(═O)R¹, —F, —Cl, —Br R⁵ = —H, Alkyl

R¹, R⁴ = —H, Alkyl. R², R³ = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl,-OAryl, —NHR¹, —NHC(═O)R¹, —F, —Cl, —Br

R¹, R⁴ = —H, Alkyl R² = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl, -OAryl,—NHR¹, —NHC(═O)R¹, —F, —Cl, —Br

R¹, R⁴ = —H, Alkyl R² = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl, -OAryl,—NHR¹, —NHC(═O)R¹, —F, —Cl, —Br

R⁴ = —H, Alkyl R² = H, -Alkyl, -Aryl, Heteroaryl, -OAlkyl, -OAryl,—NHR¹, —NHC(═O)R¹, —F, —Cl, —Br

In preferred embodiments, the diaryl-azo carboxylic acids are coupled toa minor groove binding aromatic amino acid that is3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid (CDPI)yielding DSQ derivatives of Formula III (a):

wherein Ar¹ and Ar² are aromatic or hetero-aromatic moieties, which areoptionally substituted with a functional group or a linker with afunctional group or protected functional group or a linker connecting toa synthesis solid support;X is hydroxyl, a leaving group, a linker with a functional group orprotected functional group, or a linker connecting to a synthesis solidsupport; andn is from 0 to 5.

In further preferred embodiments of the diaryl-azo carboxylic acids,with regard to Formula III (a), the Ar¹ moiety is an indole orbenzothiazole ring yielding DSQ derivatives of Formula III (b) andFormula III (c):

In additional preferred embodiments, in Formula III (b) and III (c), theAr² moiety is an aromatic or hetero-aromatic ring with an exocyclicamino group, in which said exocyclic amino group is optionallysubstituted with one or two of an alkyl, a linker with a functionalgroup or protected functional group, or a linker connecting to asynthesis solid support; n is between 0 and 3; X is —OH, a leavinggroup, a linker with a functional group or protected functional group,or a linker connecting to a synthesis solid support.

Additional preferred embodiments include Formula III derivativesrepresented by Formula III (d) and III (e):

wherein X is hydroxyl, —OPFP, or a leaving group, and n is 0, 1 or 2.

Compounds of Formula III are particularly useful for the preparation ofoligonucleotide conjugates of Formula IV (a) or IV (b):

wherein ODN is an oligonucleotide; L is a linking group having from 0 to100 main chain atoms selected from C, N, O, S, P and Si and can beacyclic, cyclic or aromatic or combinations thereof; R¹ is H, alkyl, oralkyl covalently connected to Ar³; and R³ is hydroxyl, a linking orblocking group; Ar¹ and Ar² are aromatic or hetero-aromatic moieties;and Ar³ is an aromatic moiety. If n is greater than 1, each Ar³ may bethe same or different. In preferred embodiments, the oligonucleotideconjugate may further comprise a fluorophore connected to theoligonucleotide, and the fluorophore may be any suitable fluorophireincluding FAM, AP525, AP559, AP593 or AP662. The oligonucleotide mayfurther comprise a minor groove binder connected to the oligonucleotide.The oligonucleotide conjugate may also include one or more modifiednucleobases or modified bases. The linker L may be connected to theoligonucleotide at its 3′-end or its 5′-end, or it may be connected tothe oligonucleotide at a position other than a 3′- or 5′-end.

In preferred embodiments, the oligonucleotide conjugates of Formula IV(a) are represented by Formula IV (c):

wherein n is 1, 2, or 3.

In additional preferred embodiments, the oligonucleotide conjugates ofFormula IV (c) are represented by Formula IV (d) or IV (e):

wherein n is 1, 2 or 3.

Oligonucleotide Conjugate Preparation

Oligonucleotide conjugates of the disclosure (including those of FormulaIV (a) and IV (b)) can be synthesized in a variety of ways. One way isto use the activated derivatives of Formula III, wherein X is a leavinggroup. One such leaving group is the pentafluorophenyloxy (—OPFP) groupexemplified in this disclosure. Other examples of suitable leavinggroups are N-succinimidyloxy and p-nitrophenyloxy groups as practiced inGreg T. Hermanson, Bioconjugate Techniques, Elsevier 1996, incorporatedby reference.

These derivatives can be conveniently reacted with oligonucleotidesbearing primary or secondary aliphatic amino groups to form covalentamide bonds between oligonucleotides and diaryl-azo compounds of thedisclosure. Description and examples of suitable procedures for suchconjugation reactions can be found, for instance, in Kutyavin et al.,Current Protocols in Nucleic Acid Chemistry, 2003, 8.4.1-8.4-21 andLukhtanov et al., Bioconjugate Chem. 1995, 6, 418-426.

These activated derivatives can also be used to introduce a variety offunctional groups suitable for other types of conjugation reactionsexamples of which are reviewed in Hermanson, in Bioconjugate Techniques,Elsevier, 1996, and Kolb et al., Angewandte Chemie, 2001, 40, (11),2004-2021.

A generic procedure for the preparation of diaryl-azo derivatives ofFormula III, wherein X is PFPO, is depicted in reaction Scheme 1 (FIG.2). The starting aromatic amino ester (or acid) g1 is diazotized usingnitrous or nitrosylsulfuric acid to give diazo intermediate g2, which isthen coupled with an electron-rich aromatic compound g3 to yielddiaryl-azo carboxylic ester g4 or acid g5. The obtained diaryl-azoderivatives g4 and g5 are predominantly trans-isomers in regard toorientation of the aryl groups around the azo bond. Other known methodsfor the preparation of diaryl-azo derivatives, for instance, reviewed inChem. Soc. Rev., 2011, 40, 3835-3853, can also be applied to produce therequired g5 intermediates. The diaryl-azo acid g5 is converted to PFPester g6 by reaction with pentafluorophenyl trifluoroacetate (PFP-TFA).Alternatively, using reagents and procedures known in the art, a varietyof different activated esters can be prepared starting from carboxylicacids g5. The activated PFP esters g6 are reacted with minor groovebinding aromatic amino acids (or peptides) g7 to yield carboxylic acidsg8. The amino acid and dipeptide exemplified in this specification is3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid (CDPI)and3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (CDPI₂), respectively. CDPI and CDPI₂ are fragments of known minorgrove binding agent CDPI₃ (Boger et al. J. Org. Chem., 1987, 52, 1521).Other examples of suitable g7 compounds are based on N-methylpyrrole,N-methylimidazole, N-methylhydroxypyrrole, thiazole amino carboxylicacids, building blocks of distamycin and lexitropsin, known minor groovebinding antibiotics. The carboxylic acid g8 is reacted with PFP-TFA PFPto yield the desired activated esters g9 suitable for conjugation withamine-modified oligonucleotides to yield oligonucleotide conjugates ofFormula IV (a). Alternatively, non-activated g8 acid derivatives can beconjugated to amine-modified oligonucleotides in the presence ofactivating agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC). Reagents for the preparation of amine-modified oligonucleotides,including DNA synthesis solid supports and phosphoramidites, arecommercially available.

The activated esters g9 are also used to react with linker moieties.Those linker moieties may be mono- or poly functional and containvarious functional groups such as maleimide, biotin, azide, alkyne,amine, hydroxyl, DMT-protected hydroxyl, etc. One particular type ofpoly functional linker is a 6-aminohexane-1,2-diol and is suitable forthe preparation of solid synthesis supports as illustrated in scheme 1(FIG. 2). In this approach the linker is attached to the solid supportvia a cleavable (during oligonucleotide cleavage and deprotection step)succinate linker utilizing the secondary hydroxyl group while theprimary hydroxyl and the amino groups are protected with the DMT andFMOC groups, respectively. This support is prepared in the mannerdescribed in U.S. Pat. No. 6,492,346. The FMOC group can be selectivelyremoved by treating the starting solid support with DBU yielding freeamino group available for the following reaction with PFP esters g9. Theresultant solid supports g10 are suitable for on-line oligonucleotidesynthesis starting from the DMT-protected primary hydroxyl group.Following the oligonucleotide synthesis and deprotection,oligonucleotide conjugates of Formula IV (a) are obtained. Both 3′ endand 5′ end conjugation can be achieved depending on whether 3′ or 5′nucleoside phosphoramidites are used in the synthesis. FIG. 3A shows theHPLC analysis and FIG. 3B shows the UV spectra of an oligonucleotidesynthesized using a synthesis support of type g10. This exampledemonstrates that the general approach described in scheme 1 providesthe desired oligonucleotide conjugates in good yield and the conjugatescan be readily purified by reverse phase chromatography.

FIG. 4A-4D show the structures and absorption maxima of the T8oligonucleotide conjugates synthesized according to reaction scheme 1.FIG. 4A-4D show the relationship between the DSQ structures andabsorption maxima of the T8 oligonucleotide conjugates synthesizedaccording to reaction Scheme 1. Generally, the benzothiazole-substitutedDSQ demonstrate more red-shifted absorption spectra than the phenyl orindole-substituted ones. The position of the absorption band is directlyrelated to the efficiency of FRET-based fluorescence quenching asspecified by Lakowicz, 2007.

Another example of a suitable linker for the preparation of DNAsynthesis supports of the disclosure is hydroxyprolinol, a trifunctionalreagent that has an amino, a primary and a secondary hydroxyl group.This linker as well as examples of other trifunctional reagents havingan amino, primary and a secondary hydroxyl group, are described in U.S.Pat. No. 5,512,667. The primary hydroxyl group in this example isprotected with a dimethoxytrityl group whereas the secondary hydroxyland amino groups are available for further modifications.

The synthesis support used in synthetic Scheme 1 is a highlycross-linked porous styrene-divinylbenzene copolymer furtheraminomethylated to enable the surface chemistry (Applied Biosystems, PN360865C). Another example of synthesis support is Controlled Pore Glass(CPG) (Glen Research, Sterling, Va.), which is commercially available indifferent pore sizes and with long chain alkylamine extension for moreefficient phosphoramidite coupling.

Oligonucleotide conjugates of Formula IV (a) can also be obtained usinga polystyrene synthesis support ID #480 exemplified in reaction scheme 3(FIG. 7A-7B). The synthesis supports of this type can be prepared usingreagents of general Formula III, wherein X is a linking group with aDMT-protected hydroxyl group and either Ar¹ or Ar² contain a functionalgroup for covalent attachment to a synthesis solid support via acleavable (during oligonucleotide deprotection) linker.

In the particular example depicted in reaction scheme 3, diaryl-azo-dye66 is prepared by diazotization of2-amino-1,3-benzothiazole-6-carboxylic acid followed by coupling withN-substituted aniline analog 65. The acid 66 is converted into activatedPFP ester 67 for the following condensation with CDPI₂ dipeptideyielding intermediate 68. This intermediate is activated via theformation of PFP ester 69 and then reacted with O-DMT-protectedaminohexanol to afford intermediate 70 with two orthogonally-protectedhydroxyl groups. The acetyl protection of the intermediate 70 isselectively saponified to give hydroxyl intermediate 71. To introducethe cleavable succinate linker, hydroxyl intermediate 71 is reacted withsuccinic anhydride and then activated with PFP-TFA. The obtained PFPester 72 is reacted with aminomethyl polystyrene support (e.g. used insynthetic scheme 1) to yield synthesis support ID#480. Analytical HPLCis shown in FIG. 8A and PDA UV-VIS spectra is shown in FIG. 8B for anexample of oligonucleotide synthesis using the synthesis support ID#480.It demonstrates that the DSQ ID#480-modified oligonucleotide conjugatescan be synthesized in excellent yield and be readily purified by reversephase chromatography.

Some oligonucleotide conjugates of Formula IV (b) can be prepared usingthe synthesis solid support ID #478 prepared according to the reactionscheme 2 (FIG. 5A-5B). The approach depicted in this scheme requires anintroduction of a hydroxyl group, starting point for oligonucleotidesynthesis, into the diaryl-azo section of compounds of Formula I. Thisis achieved in the first step of the reaction scheme by using6-[methyl(phenyl)amino]hexyl acetate (WO 2008008481), an aniline analogwith a protected 6-hydroxyhexyl linker. This aniline is coupled withethyl 2-diazo-1,3-benzothiazole-6-carboxylate to yield diaryl-azo dye60. The bis-ester 60 is then saponified to give hydroxyl acid 61. Thehydroxyl group is then DMT-protected followed by activation with PFP-TFAto afford PFP ester 62. Condensation of the ester with CDPI₂ producedintermediate 63, which then was activated with PFP-TFA to afford thedesired PFP ester 64, suitable for the following solid supportchemistry. The starting N-MMT-5-aminopentylglycolate polystyrene support(prepared according to U.S. Pat. No. 7,759,126) was first deprotectedwith trichloroacetic acid in dichloromethane and then reacted with thePFP ester 64 to give polystyrene synthesis support ID #478. Ananalytical C18 HPLC trace of a T8 oligonucleotide synthesis using thissupport is shown in FIG. 6A, with PDA UV-VIS spectra of the main peakand product structure shown in FIG. 6B. This example demonstrates thatthe oligonucleotide conjugates prepared according to scheme 2 can besynthesized in excellent yield and be readily purified by reverse phasechromatography.

2-Cyanoethyl N,N-diisopropylphosphoramidite chemistry is another way toprepare oligonucleotides bearing the compounds of the disclosure. Oneexample of a suitable phosphoramidite reagent is shown in reactionscheme 4 (FIG. 9A-9B). In this approach, the intermediate 70 (reactionscheme 3) is first reacted with diethylpyrocarbonate to protect theindole and amide NH groups yielding intermediate 73. The DMT group isthen selectively removed and the resultant alcohol 74 is converted intothe phosphoramidite 75 by reaction with 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite. FIG. 10A shows an analyticalC18 HPLC trace of a T8 oligonucleotide synthesis using thisphosphoramidite, and FIG. 10B shows PDA UV-VIS spectra of the main peakand product structure. This demonstrates that the oligonucleotideconjugates prepared using the phosphoramidite 75 can be synthesized in afair yield and be purified by reverse phase chromatography. Furtherimprovement in purity in efficiency of the phosphoramidite approach canbe achieved through the use of N-substituted aminohexanol in scheme 3(e.g. N-methyl-6-aminohexanol as described in U.S. Pat. No. 9,056,887)by eliminating the formation of the imidazolidine-2,4-dione side-productshown in scheme 4 (FIG. 9A-9B).

FIG. 11A-11C show UV-VIS absorption spectra of T8-DSQ conjugates. Theabsorption bands of these exemplary compounds cover the wavelength rangebetween 400 and 700 nm, which is suitable for efficient FRET quenchingof common fluorophores for real-time PCR applications.

Applications of Oligonucleotide Conjugates

Oligonucleotide conjugates of Formula IV (a) and (b) are useful forvarious application as described below. Preferred embodiments include amethod for detecting a target nucleic acid sequence in a sample,comprising contacting the sample with embodiments of the oligonucleotideconjugates described herein, wherein the oligonucleotide conjugate has anucleic acid sequence at least partially complementary to the targetnucleic acid sequence, and wherein the oligonucleotide conjugate furthercomprises a fluorophore, and detecting a fluorescent signal from theoligonucleotide conjugate upon hybridization to the target nucleic acidsequence. Further preferred embodiments also include the step ofamplifying the target nucleic acid sequence. In some preferredembodiments the oligonucleotide conjugate is a primer or a probe.

In one embodiment of Formula IV the DSQ-conjugates comprise a 3′-DSQ anda 5′-fluorophore to yield oligonucleotides of general Formula V:

5′-Fl-ODN-DSQ   Formula V

wherein Fl is a fluorophore and ODN-DSQ is an oligonucleotide conjugateof Formula IV (a) or (b).

Conjugates of Formula V are particularly useful as DNA probes forfluorescence-based real-time and endpoint PCR applications that rely onthe hybridization-dependent 5′-exonuclease activity of DNA polymerasefor fluorophore cleavage, also known as the TaqMan technology. Examplesof TaqMan PCR applications using conjugates of Formula V are shown inFIG. 16.

In other preferred embodiments, with reference to Formula IV, theoligonucleotide conjugates comprise a 5′ DSQ and a 3′ fluorophore asrepresented by Formula VI:

5′-DSQ-ODN-Fl   Formula VI

wherein Fl is a fluorophore and DSQ-ODN is an oligonucleotide conjugateof Formula IV (a) or (b).

Oligonucleotide conjugates of Formula VI are useful as DNA probes forfluorogenic probes whose fluorescence signal is generated due to thehybridization with a target. The 5′ DSQ moiety prevents the 5′exonuclease probe degradation. One particular type of such fluorogenicprobes is the Molecular Beacon technology, which relies on the stem-loopstructure to improve signal-to-background ratio and mismatchdiscrimination.

In additional preferred embodiments, with reference to Formula IV, theoligonucleotide conjugates comprise a 5′-DSQ and a 5′-fluorophorepositioned adjacent to each other as represented by Formula VII:

5′-DSQ-Fl-ODN   Formula VII

Such oligonucleotides are useful as hybridization-triggered fluorogenicprobes and primers as illustrated in FIG. 14A-14B and Table 14.

In additional preferred embodiments, the oligonucleotides of Formula IVcould be used in digital PCR and arrays (U.S. Pat. Nos. 9,328,384 and7,759,126 incorporated by reference).

Although not illustrated in Formulas V-VII, in some preferredembodiments the DSQ moiety is covalently attached to an internalposition of an oligonucleotide, for example to an amine-tailednucleobase.

In further preferred embodiments, the DSQ oligonucleotides are used todifferentiate single nucleotide polymorphisms as taught in U.S. Pat. No.6,312,894 and, 7,718,374 incorporated by reference. In a relatedembodiment, different targets are detected utilizing melting curveanalyse as taught in Hymas and Hillyard, 2009, incorporated byreference.

Other applications, not specifically described in this specification butknown in the art (for example Didenko, Biotechniques 2001, 31(5):1106-1121, Kim, Y. et al., Int. J. Clin. Exp. Pathol. (2008) 1, 105-116,and U.S. Pat. No. 7,790,385), are also potential applications of the newderivatives and oligonucleotides of the disclosure.

EXAMPLES

The following examples illustrate the functional performance ofoligonucleotides labeled with preferred embodiments of the DSQderivatives and are not intended to limit the scope of the claims.

Example 1. Preparation of DSQ Intermediates and PFP Esters Ethyl5-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1H-indole-2-carboxylate (1)

To a suspension of ethyl 5-aminoindole-2-carboxylate (3.0 g, 14.7 mmol)in 80 mL of water was added 2.5 mL of conc. HCl. The stirred suspensionwas cooled in ice/water bath to 0-4° C. and treated with a solution ofNaNO₂ (1.1 g, 15.9 mmol) in 20 mL of water via an addition funnel over 5min. The reaction was stirred at 0-4° C. for 20 min then warmed to roomtemperature and stirred for another 10 min to give a dark, mostly clearsolution. N,N-Dimethylaniline (2.5 mL, 19.7 mmol) was added in oneportion and stirring was continued for 1 h. The precipitated solid wascollected by filtration, washed with water and resuspended in 50 mL ofMeOH. The suspension was heated to reflux then cooled and filtered tocollect the precipitated solid. Drying in vacuo afforded 2.9 g (59%yield) of the desired dye 1 as a brown-orange solid. ¹H NMR (DMSO-d6) δ12.17 (s, 1H), 8.14 (d, J=1.8 Hz, 1H), 7.83 (dd, J₁=9 Hz, J₂=2.1 Hz,1H), 7.79 (d, J=9 Hz, 2H), 7.53 (d, J=8.7 Hz, 1H), 7.31 (d, J=0.9 Hz,1H), 6.84 (d, J=8.7 Hz, 2H), 4.37 (q, J=7.2 Hz, 2H), 3.05 (s, 6H), 1.36(t, J=7.2 Hz, 3H).

5-{(E)-[4-(Dimethylamino)phenyl]diazenyl}-1H-indole-2-carboxylic Acid(2)

A suspension of 1 (2.9 g, 8.6 mmol) in a mixture of THF (60 mL), MeOH(40 mL) and 1N NaOH (20 mL) was heated at 50° C. with stirring for 2.5 hto give a clear red-brown solution. The reaction was cooled, neutralizedwith 20 mL of 1N HCl and concentrated to about 40 mL. The precipitatedblack solid was collected by filtration, washed with water and dried toafford 2.6 g (98% yield) of acid 2 as a black solid. ¹H NMR (DMSO-d6) δ12.06 (s, 1H), 8.13 (d, J=1.5 Hz, 1H), 7.83 (dd, J₁=9 Hz, J₂=1.8 Hz,1H), 7.78 (d, J=9 Hz, 2H), 7.52 (d, J=8.7 Hz, 1H), 7.26 (d, J=1.5 Hz,1H), 6.84 (d, J=8.7 Hz, 2H), 3.05 (s, 6H).

Pentafluorophenyl5-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1H-indole-2-carboxylate (3)

To a suspension of 2 (2.6 g, 8.4 mmol) in 30 mL of anhydrous CH₂Cl₂ wasadded triethylamine (4.2 mL) followed by pentafluorophenyltrifluoroacetate (PFP-TFA) in several portions until no more startingacid 2 was observed by TLC or HPLC analysis. Total of 3.5 mL (20.25mmol) of PFP TFA was added over 16 h. The completed reaction wasconcentrated and the obtained residue resuspended in cold MeOH (50 mL).The insoluble material was collected by filtration, washed with MeOH(2×10 mL) and dried to afford 2.7 g (68% yield) of 3 as a brown-orangesolid. ¹H NMR (DMSO-d6) δ 12.72 (s, 1H), 8.22 (d, J=1.5 Hz, 1H), 7.93(dd, J₁=8.7 Hz, J₂=1.8 Hz, 1H), 7.80 (d, J=9 Hz, 2H), 7.73 (d, J=1.5 Hz,1H), 7.61 (d, J=9 Hz, 1H), 6.84 (d, J=9 Hz, 2H), s (3.05, 6H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (4)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (WO 2004043350) (2.5 mmol) in 10 mL of anhydrous dimethylformamide(DMF) was added triethylamine (1.5 mL) followed by 1.2 g (2.5 mmol) ofPFP ester 3. The reaction was briefly stirred to initially give a clearsolution but the product precipitation started within minutes. Thereaction was agitated for 20 h then the precipitated material wascollected by filtration, washed with small amount of DMF (2 mL), MeOH(10 mL) and ether. Drying in vacuo afforded 1.6 g (94% yield) of thetitle acid 4 (partial triethylammonium salt) as an orange-brown solid.¹H NMR (DMSO-d6) δ 12.00 (s, 1H), 11.78 (s, 1H), 11.02 (s, 1H), 8.26 (m,2H), 8.16 (d, J=1.5 Hz, 1H), 7.81 (dd, J₁=8.7 Hz, J₂=1.8 Hz, 1H), 7.80(d, J=9 Hz, 2H), 7.58 (d, 9 Hz, 1H), 7.39 (d, 9 Hz, 1H), 7.32 (m, 2H),7.12 (d, J=1.5 Hz, 1H), 6.93 (d, J=1.8 Hz, 1H) 6.85 (d, J=9 Hz, 2H),4.68 (m, 4H), 3.4 (m, 6H), 3.06 (s, 6H), 2.76 (m, 4H), 1.06 (t, J=7.2Hz, 6H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(5)

To a suspension of 5 (1.5 g, 2.2 mmol) in 10 mL of anhydrous DMF wasadded 1.2 ml of triethylamine followed by several portions of PFP-TFAover the course of 24 h until no more starting 5 was found by HPLCanalysis. A total of 0.9 mL (5.2 mmol) of PFP-TFA was used. MeOH (20 mL)was added to initiate product precipitation. The resultant solid wascollected by filtration, washed with methanol (2×10 mL) and ether.Drying in vacuo afforded 1.73 g (93% yield) of PFP ester 5 as a brownsolid. NMR analysis indicated a partial (40%) TFA-protection of theindole NH-groups. ¹H NMR (DMSO-d6) δ 12.64 (s, 0.4H), 12.55 (s, 0.6H),12.07 (s, 0.4H), 12.00 (s, 0.6H), 11.81 (s, 0.6H), 8.44 (m, 1H), 8.29(m, 1H), 8.16 (s, 1H), 7.8 (m, 3H), 7.7-7.1 (m, 6H), 6.85 (d, J=9 Hz,2H), 4.72 (m, 4H), 3.5 (m, 4H), 3.06 (s, 6H).

Ethyl5-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1H-indole-2-carboxylate(6)

To a suspension of ethyl 5-aminoindole-2-carboxylate (3.0 g, 14.7 mmol)in 80 mL of water was added 2.5 mL of conc. HCl. The stirred suspensionwas cooled in ice/water bath to 0-4° C. and treated with a solution ofNaNO₂ (1.1 g, 15.9 mmol) in 20 mL of water via an addition funnel overthe course of 5 min. The reaction was stirred at 0-4° C. for 20 min thenwarmed to room temperature and stirred for another 10 min to give adark, mostly clear solution. 1-(2,5-dimethoxyphenyl)pyrrolidine (M.Sarma et al. J. Org. Chem. 2012, 77(1), 432-444) (4.08 g, 19.7 mmol) wasadded in one portion and stirring was continued for 10 min before solidsodium acetate trihydrate (5.3 g) was added. The resultant brownsuspension was stirred for 2 h. The precipitated solid was collected byfiltration, washed with water, MeOH and dried in vacuo to afford 5.3 g(85% yield) of the desired dye 6 as a brown-orange solid. ¹H NMR(DMSO-d6) δ 12.11 (s, 1H), 8.08 (d, J=1.5 Hz, 1H), 7.78 (dd, J₁=9 Hz,J₂=1.8 Hz, 1H), 7.50 (d, J=9 Hz, 2H), 7.33 (s, 1H), 7.29 (d, J=1.2 Hz,1H), 6.30 (s, 1H), 4.37 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.75 (s, 3H),3.51 (m, 4H), 1.90 (m, 4H), 1.36 (t, J=7.2 Hz, 3H).

5-{(E)-[2,5-Dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1H-indole-2-carboxylicAcid (7)

To a solution of 6 (5.0 g, 11.8 mmol) in 90 mL of THF was added 60 mL ofMeOH and 30 mL of 1N NaOH. The initial suspension was stirred at 50° C.for 1.5 h to give a dark solution. The reaction was cooled, neutralizedwith 1N HCl (30 mL) and concentrated to about 60 mL. The resultant solidwas collected by filtration, washed with water and dried to give 4.5 g(97% yield) of acid 7 as a black solid. ¹H NMR (DMSO-d6) δ 13.06 (br s,1H), 12.01 (s, 1H), 8.06 (d, J=1.5 Hz, 1H), 7.76 (dd, J₁=9 Hz, J₂=1.2Hz, 1H), 7.49 (d, J=8.7 Hz, 2H), 7.35 (s, 1H), 7.23 (s, 1H), 6.29 (s,1H), 3.95 (s, 3H), 3.76 (s, 3H), 3.54 (br s, 4H), 1.89 (br s, 4H).

Pentafluorophenyl5-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1H-indole-2-carboxylate(8)

A solution of 7 (1.18 g, 3.0 mmol) and triethylamine (2.2 mL) in 20 mLof anhydrous CH₂Cl₂ was added PFP-TFA in several portions over thecourse of 2 days to a total amount of 1.76 mL (10.1 mmol). The reaction(suspension of partially precipitated product) was concentrated andresuspended in 20 mL of MeOH. The insoluble material was collected byfiltration, washed with MeOH and dried in vacuo to afford 1.33 g (79%yield) of PFP ester 8 as an orange solid. ¹H NMR (DMSO-d6) δ 12.67 (s,1H), 8.16 (d, J=1.5 Hz, 1H), 7.89 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.73 (d,J=1.2 Hz, 1H), 7.58 (d, J=9 Hz, 2H), 7.35 (s, 1H), 6.30 (s, 1H), 3.95(s, 3H), 3.75 (s, 3H), 3.52 (m, 4H), 1.90 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (9)

To a solution3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.8 mmol) in 4 mL of anhydrous DMF was added triethylamine (0.5mL) followed by 0.45 g (0.8 mmol) of PFP ester 8. The initial suspensionwas stirred at room temperature for 5 min and then briefly warmed up toapprox. 45° C. to give a clear red solution. Product precipitationstarted soon after that. The reaction was allowed to proceed for 20 h.The precipitated material was collected by filtration, washed with DMF(2×2 ml), acetone (2×10 mL) and dried in vacuo to give 0.56 g (96%yield) of acid 9 (partial triethylammonium salt) as a brown solid. ¹HNMR (DMSO-d6) δ 11.95 (s, 1H), 11.77 (s, 1H), 11.61 (s, 1H), 8.26 (m,2H), 8.11 (d, J=1.2 Hz, 1H), 7.76 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.55 (d,J=8.7 Hz, 2H), 7.38 (d, 9 Hz, 1H), 7.35 (s, 1H), 7.32 (m, 2H), 7.12 (d,J=1.5 Hz, 1H), 6.92 (s, 1H) 6.32 (s, 1H), 4.68 (m, 4H), 3.95 (s, 3H),3.76 (s, 3H), 3.5-3.3 (m, 8H), 2.67 (q, J=7.2 Hz, 4H), 1.91 (m, 4H),1.06 (t, J=7.2 Hz, 6H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(10)

To a suspension of 9 (0.47 g, 0.62 mmol) in 6 mL of anhydrous DMF wasadded 0.5 mL of triethylamine and 0.2 mL (1.16 mmol) of PFP-TFA. Thereaction was stirred for 8 h and treated with another portion of PFP-TFA(0.05 mL) to complete the PFP ester formation. After being stirred for16 h the precipitated material was collected by filtration, washed withDMF (2×1 mL), acetone (4×5 mL) and dried in vacuo to give 0.43 (75%yield) of PFP ester 10 as an orange solid. ¹H NMR (DMSO-d6) δ 12.55 (s,1H), 11.95 (s, 1H), 11.80 (s, 1H), 8.42 (br d, J=9 Hz, 1H), 8.29 (br d,J=8 Hz, 1H), 8.10 (d, J=1.2 Hz, 1H), 7.76 (dd, J₁=9 Hz, J₂=1.5 Hz, 1H),7.60 (d, J=1.5 Hz, 1H), 7.55 (d, J=9 Hz, 2H), 7.43 (d, 9 Hz, 1H), 7.39(d, 9 Hz, 1H), 7.35 (s, 1H), 7.30 (s, 1H), 7.16 (s, 1H) 6.32 (s, 1H),4.71 (m, 4H), 3.95 (s, 3H), 3.76 (s, 3H), 3.5 (m, 8H), 1.91 (m, 4H).

Ethyl2-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylate(11)

Ethyl 2-amino-1,3-benzothiazole-6-carboxylate (4.44 g, 20 mmol) wasresuspended in 100 mL of a 1:1 mixture of acetic and propionic acids.The partial solution was cooled to 0-4° C. and treated with 6.7 mL of40% nitrosylsulfuric acid maintaining the temperature below 10° C. Thereaction was stirred for 5 h then allowed to briefly warm to roomtemperature before cooling again to 0-4° C. The obtained solution ofdiazonium salt was added via an addition funnel to a stirred cold(ice/water bath) mixture prepared by combining a solution ofN,N-dimethylaniline (3 mL) in 25 mL of MeOH and a solution of 0.6 g ofsulfamic acid in 200 mL of water. The stirring was continued for 3 hbefore the reaction was neutralized by adding approx. 200 mL of 40%aqueous trimethylamine to a pH of 6-7. The resultant solid was collectedby filtration, washed with water and resuspended in hot MeOH (50 mL).After cooling the insoluble material was collected by filtration, washedwith MeOH (2×10 mL) and dried in vacuo to afford 4.34 g (61% yield) ofdye 11 as a dark-purple solid. ¹H NMR (CDCl₃) δ 8.55 (d, J=1.5 Hz, 1H),8.14 (skewed dd, J₁=8.4 Hz, J₂=1.5 Hz, 1H), 8.07 (skewed d, J=8.4 Hz,1H), 8.02 (d, J=9.6 Hz, 2H), 6.78 (d, J=9.6 Hz, 2H), 4.44 (q, J=7.2 Hz,2H), 3.19 (s, 6H), 1.43 (t, J=7.2 Hz, 3H).

2-{(E)-[4-(Dimethylamino)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylicAcid (12)

To a solution of 11 (0.18 g, 0.51 mmol) in 9 mL of THF was added MeOH (6mL) followed by 3 mL of 1N NaOH. The mixture was stirred at 50° C. for70 min, cooled, neutralized with 1N HCl (3 mL) and concentrated to about5 mL. The precipitated material was collected by filtration, washed withwater and dried to afford 0.143 g (86% yield) of the desired acid 12 asa black solid. ¹H NMR (DMSO-d6) δ 13.10 (br s, 1H), 8.60 (s, 1H), 8.02(s, 2H), 7.87 (d, J=9 Hz, 2H), 6.93 (d, J=9 Hz, 2H), 3.18 (s, 6H).

Pentafluorophenyl2-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylate(13)

To a solution of 12 (0.14 g, 0.42 mmol) and triethylamine (0.4 mL) in 2mL of anhydrous DMF was added 0.2 mL (1.16 mmol) of PFP-TFA. After beingstirred for 30 min the reaction was concentrated and diluted withacetone (4 mL). The precipitated material was collected by filtrationwashed with acetone (2 mL), 20% ethyl acetate/hexane (5 mL) and dried invacuo to give 0.158 g (76% yield) of PFP ester 13 as a dark brown solid.¹H NMR (CDCl₃) δ 8.72 (d, J=1.5 Hz, 1H), 8.26 (skewed dd, J₁=8.4 Hz,J₂=1.5 Hz, 1H), 8.16 (skewed d, J=8.4 Hz, 1H), 8.03 (d, J=9.3 Hz, 2H),6.79 (d, J=9.6 Hz, 2H), 3.21 (s, 6H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(2-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (14)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.33 mmol) in 5 mL of anhydrous DMF was added triethylamine (0.2mL) followed by 0.156 g (0.32 mmol) of PFP ester 13. The reaction wasstirred at 50° C. for 5 h and then cooled to room temperature. Theprecipitated material was collected by filtration, washed with DMF (2×1ml), acetone (2×5 mL) and dried in vacuo to give 0.167 g (75% yield) ofacid 14 (partial triethylammonium salt) as a brown solid. ¹H NMR(DMSO-d6) δ 11.75 (s, 1H), 11.66 (s, 1H), 8.30 (s, 1H), 8.1-8.3 (m, 2H),8.05 (d, J=8.4 Hz, 1H), 7.88 (d, J=9.6 Hz, 2H), 7.72 (d, J=8.1 Hz, 1H),7.35 (br s, 1H), 7.29 (d, J=9 Hz, 1H), 7.07 (s, 1H), 6.94 (d, J=9 Hz,2H), 6.93 (s, 1H), 4.62 (t, J=8.7 Hz, 2H), 4.20 (t, J=8.4 Hz, 2H), 3.3(m, 8H), 3.18 (s, 6H), 2.68 (m, 3H), 1.02 (t, J=7 Hz, 4H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(2-{(E)-[4-(dimethylamino)phenyl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(15)

To a suspension of 14 (0.163 g, 0.23 mmol) in 2 mL of anhydrous DMF wasadded 0.1 mL of triethylamine and 0.1 mL (0.58 mmol) of PFP-TFA. Afterbeing stirred for 16 h the reaction was concentrated and the resultingmaterial resuspended in acetone (2-3 mL). Filtration, wash with acetoneand drying in vacuo afforded 0.175 g (88% yield) of PFP ester 15 as abrown solid. NMR analysis indicated a partial (25%) TFA-protection ofthe indole NH-groups. ¹H NMR (DMSO-d6) δ 12.62 (s, 0.25H), 12.52 (s,0.75H), 11.78 (s, 0.75H), 8.5-8.1 (m, 3H), 8.06 (d, J=8.7 Hz, 0.25H),8.05 (d, J=8.7 Hz, 0.75H), 7.87 (d, J=9 Hz, 2H), 7.73 (m, 1H), 7.61 (d,J=1.2 Hz, 0.25H), 7.57 (d, J=1.5 Hz, 0.75H), 7.5-7.3 (m, 2H), 7.09 (s,1H), 6.94 (d, J=9 Hz, 2H), 4.66 (m, 2H), 4.20 (m, 2H), 3.46 (m, 2H),3.29 (m, 2H), 3.17 (s, 6H).

Ethyl2-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylate(16)

To a suspension of ethyl 2-amino-1,3-benzothiazole-6-carboxylate (0.64g, 2.86 mmol) in acetic acid (8 mL) was added 1 mL of 40%nitrosylsulfuric acid with stirring over a period of 1 min to give alight yellow partial solution. The mixture was stirred at roomtemperature for 30 min before being used in the next step. A solution of1-(2,5-dimethoxyphenyl)pyrrolidine (0.88 g, 4.2 mmol) in 20 mL of THFwas combined with a solution of sodium acetate trihydrate (2.4 g) in 12mL of water to give a biphasic mixture. The mixture was cooled to 0-4°C. and treated with the solution of diazonium salt from step 1. Theresulting blue suspension was stirred for 1 h and then poured intosaturated NaHCO₃. The crude dye was extracted with ethyl acetate andpurified on silica eluting with a gradient of ethyl acetate in hexane.Concentration of the pure product fractions afforded 0.17 g (13% yield)of dye 16 as a dark purple solid. ¹H NMR (DMSO-d6) δ 8.54 (d, J=1.8 Hz,1H), 7.99 (skewed dd, J₁=8.7 Hz, J₂=1.8 Hz, 1H), 7.89 (skewed d, J=8.4Hz, 1H), 7.31 (s, 1H), 6.20 (s, 1H), 4.35 (q, J=7.2 Hz, 2H), 4.00 (s,3H), 3.81 (s, 3H), 3.79 (m, 4H), 1.95 (m, 4H), 1.36 (t, J=7.2 Hz, 3H).

2-{(E)-[2,5-Dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylicAcid (17)

To a solution of 11 (0.17 g, 0.39 mmol) in 9 mL of THF was added MeOH (6mL) followed by 3 mL of 1N NaOH. The mixture was stirred at 50° C. for70 min, cooled, neutralized with 1N HCl (3 mL) and concentrated to about5 mL. The precipitated material was collected by filtration, washed withwater and dried to afford 0.16 g (99% yield) of the desired acid 17 as ablack solid. ¹H NMR (DMSO-d6) δ 8.51 (d, J=1.5 Hz, 1H), 7.98 (skewed dd,J₁=8.4 Hz, J₂=1.5 Hz, 1H), 7.82 (skewed d, J=8.4 Hz, 1H), 7.28 (s, 1H),6.21 (s, 1H), 4.01 (s, 3H), 3.85 (m, 4H), 3.83 (s, 3H), 1.96 (m, 4H).

Pentafluorophenyl2-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1,3-benzothiazole-6-carboxylate(18)

A solution of 17 (0.16 g, 0.39 mmol) and triethylamine (0.4 mL) in 2 mLof anhydrous DMF was treated with several portions (0.05 mL each) ofPFP-TFA until no more starting 17 was found by HPLC analysis. A total of0.35 mL (2 mmol) of PFP-TFA was used. The reaction was concentrated andchromatographed on silica eluting with a gradient of ethyl acetate(50-100%) in hexane. Concentration of the pure product fractionsafforded 0.14 g (62% yield) of PFP ester 18 as a black solid. ¹H NMR(CDCl₃) δ 8.51 (m, J=0.9 Hz, 1H), 8.21 (skewed d of m, J₁=8.4 Hz, J₂=0.9Hz, 1H), 8.03 (skewed d, J=8.4 Hz, 1H), 7.55 (s, 1H), 6.01 (s, 1H), 4.05(s, 3H), 3.82 (s, 3H), 3.77 (m, 4H), 2.01 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(2-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (19)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.245 mmol) in 5 mL of anhydrous DMF was added triethylamine (0.2mL) followed by 0.14 g (0.24 mmol) of PFP ester 18. The reaction wasstirred at 50° C. for 3 h and then cooled to room temperature. Theprecipitated material was collected by filtration washed with acetone(3×5 mL) and dried in vacuo to give 0.117 g (62% yield) of acid 19(partial triethylammonium salt) as a black solid. ¹H NMR (DMSO-d6) δ11.78 (s, 1H), 11.71 (s, 1H), 8.4-8.0 (m, 3H), 7.93 (d, J=8.4 Hz, 1H),7.68 (d, J=8.1 Hz, 1H), 7.33 (s, 1H), 7.4-7.2 (m, 2H), 7.10 (s, 1H),6.98 (s, 1H), 6.21 (s, 1H), 4.66 (t, J=8.1 Hz, 2H), 4.23 (t, J=8.1 Hz,2H), 4.00 (s, 3H), 3.82 (s, (3H), 3.78 (m, 4H), 3.45 (m, 2H), 2.71 (m,2H), 1.96 (m, 4H), 1.04 (t, J=8.4 Hz, 3H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(2-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(20)

To a suspension of 19 (0.117 g, 0.15 mmol) in 2 mL of anhydrous DMF wasadded triethylamine (0.1 mL) followed by PFP-TFA (0.1 mL, 0.58 mmol).The reaction was stirred for 1 h and then concentrated. The resultantmaterial was resuspended in acetone (3 mL) and filtered. The collectsolid was washed with acetone and dried in vacuo to afford 0.125 g (88%yield) of compound 20 as a dark-purple solid. ¹H NMR (DMSO-d6) δ 12.57(s, 1H), 11.82 (s, 1H), 8.44 (d, J=7.8 Hz, 1H), 8.4-8.0 (m, 2H), 7.92(d, J=8.4 Hz, 1H), 7.8-7.6 (m, 3H), 7.5-7.2 (m, 3H), 7.12 (s, 1H), 6.19(s, 1H), 4.70 (t, J=8.1 Hz, 2H), 4.23 (t, J=8.1 Hz, 2H), 4.00 (s, 3H),3.82 (s, (3H), 3.78 (m, 4H), 3.5-3.3 (m, 4H), 1.96 (m, 4H).

Methyl3-chloro-4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}benzoate(21)

To a suspension of methyl 4-amino-3-chlorobenzoate (1.1 g, 5.9 mmol) in32 mL of water was added 1 mL of conc. HCl. The stirred suspension wascooled in ice/water bath to 0-4° C. and treated with a solution of NaNO₂(0.44 g, 6.3 mmol) in 8 mL of water via an addition funnel over thecourse of 5 min. The reaction was stirred at 0-4° C. for 30 min thenwarmed to room temperature and stirred for another 10 min to give amostly complete solution. 1-(2,5-dimethoxyphenyl)pyrrolidine (1.9 g, 9.1mmol) was added in one portion and stirring was continued for 10 minbefore solid sodium acetate trihydrate (5.0 g) was added. The reactionwas allowed to warm to room temperature, stirred for 3 h and thencarefully neutralized with saturated NaHCO₃. The precipitated solid wascollected by filtration, washed with water and dried in vacuo. The crudeproduct was crystallized from hot MeOH to afford 1.8 g (67% yield) ofdye 21 as a black crystalline solid. ¹H NMR (CDCl₃) δ 8.14 (d, J=1.8 Hz,1H), 7.92 (dd, J₁=8.7 Hz, J₂=1.8 Hz, 1H), 7.71 (d, J=8.7 Hz, 1H), 7.50(s, 1H), 6.14 (s, 1H), 4.01 (s, 3H), 3.93 (s, 3H), 3.82 (s, 3H), 3.63(m, 4H), 1.98 (m, 4H).

3-Chloro-4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}benzoicAcid (22)

A solution of 21 (1.6 g, 3.96 mmol) in a mixture of THF (90 mL) and MeOH(60 mL) was treated with 1N NaOH (30 mL). The reaction was heated at 50°C. with stirring for 2.5 h to give a clear red solution. The reactionwas cooled, neutralized with 30 mL of 1N HCl and concentrated to about60 mL. The precipitated solid was collected by filtration, washed withwater and dried in vacuo to afford 1.7 g (98% yield) of acid 22 as ablack solid. ¹H NMR (DMSO-d6) δ 12.15 (br s, 1H), 8.02 (s, 1H), 7.93 (d,J=7.5 Hz, 1H), 7.65 (d, J=7.8 Hz, 1H), 7.34 (s, 1H), 6.22 (s, 1H), 3.97(s, 3H), 3.77 (s, 3H), 3.68 (m, 4H), 1.93 (m, 4H).

Pentafluorophenyl3-chloro-4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}benzoate(23)

Acid 22 (1.7 g, 4.3 mmol) was dried by co-evaporation with 25 mL ofanhydrous DMF and 1 mL of triethylamine. The dried material wasre-dissolved in another portion (25 mL) of DMF with 1 mL oftriethylamine and treated with 0.75 mL (4.3 mmol) of PFP-TFA. Thereaction was stirred for 60 min to give a thick suspension due to theproduct precipitation. DMF was removed in vacuo and the resultant solidresuspended in water. The precipitate was collected by filtration,washed with MeOH and dried in vacuo over P₂O₅. The crude PFP ester 23was re-crystallized from ethyl acetate to afford 1.94 g (81% yield) ofPFP ester 23 as a black crystalline solid. ¹H NMR (CDCl₃) δ 8.29 (d,J=1.8 Hz, 1H), 8.06 (dd, J₁=8.7 Hz, J₂=1.8 Hz, 1H), 7.82 (d, J=8.7 Hz,1H), 7.52 (s, 1H), 6.11 (s, 1H), 4.02 (s, 3H), 3.83 (s, 3H), 3.67 (m,4H), 1.99 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[3-chloro-4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}benzoyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (24)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.8 mmol) in 4 mL of anhydrous DMF was added triethylamine (0.5mL) followed by 0.44 g (0.8 mmol) of PFP ester 23. The reaction wasstirred at 50° C. for 5 h and then cooled to room temperature. Theprecipitated material was collected by filtration, washed with DMF (3×2ml), acetone (2×5 mL) and dried in vacuo to give 0.46 g (75% yield) ofacid 24 (partial triethylammonium salt) as a brown solid. ¹H NMR(DMSO-d6) δ 11.79 (s, 1H), 11.69 (s, 1H), 8.3-8.1 (m, 2H), 7.84 (s, 1H),7.66 (apparent br s, 2H), 7.4-7.2 (m, 3H), 7.10 (s, 1H), 6.97 (s, 1H),6.27 (s, 1H), 4.65 (t, J=7.5 Hz, 2H), 4.22 (t, J=7.8 Hz, 2H), 3.97 (s,3H), 3.76 (s, 3H), 3.62 (m, 4H), 3.5-3.3 (m, 4H), 2.73 (m, 3H), 1.93 (m,4H), 1.05 (t, J=7.2 Hz, 4H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[3-chloro-4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}benzoyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(25)

To a suspension of 24 (0.46 g, 0.61 mmol) in 6 mL of anhydrous DMF wasadded 0.3 ml of triethylamine followed by several portions of PFP-TFAover the course of 24 h until no more starting 24 was found by HPLCanalysis. A total of 0.36 mL (2.1 mmol) of PFP-TFA was used. Thereaction was concentrated and the residue resuspended in acetone. Theprecipitate was collected by filtration, washed with acetone and driedin vacuo to give 0.44 g (78% yield) of PFP ester 25 as a dark brownsolid. ¹H NMR (DMSO-d6) δ 12.56 (s, 1H), 11.82 (s, 1H), 8.44 (d, J=8.4Hz, 1H), 8.3-8.1 (m, 2H), 7.84 (s, 1H), 7.66 (apparent br s, 2H), 7.6(s, 1H), 7.4 (m, 3H), 7.12 (s, 1H), 6.22 (s, 1H), 4.69 (t, J=7.5 Hz,2H), 4.22 (t, J=7.8 Hz, 2H), 3.97 (s, 3H), 3.76 (s, 3H), 3.63 (m, 4H),3.6-3.3 (m, 4H), 1.93 (m, 4H), 3.49 (t, 2H), 3.30 (m, 2H), 1.93 (m, 4H).

4-[(E)-(4-Amino-2,5-dimethoxyphenyl)diazenyl]benzoic Acid (26)

A solution of NaNO₂ (2.5 g, 36.5 mmol) in 25 mL of water was added viaan addition funnel over the course of 40 min to a cold (0-4° C.) stirredsolution of 4-aminobenzoic acid in a mixture of water (200 mL) and conc.HCl (6.2 mL). The resulting light yellow solution was stirred at 0-4° C.for 30 min before being used in the next step. Solid2,5-dimethoxyaniline (5.59 g, 36.5 mmol) was added in one portion to thestirred solution of diazonium salt from step 1. The reaction was stirredat 0-4° C. for 1.5 h and then at room temperature for 1 h to give asuspension due to the product precipitation. The precipitate wascollected by filtration, washed with water and dried. The crude dye waschromatographed on silica eluting with a gradient of MeOH in CH₂Cl₂(0-20%). The pure dye fractions were concentrated to afford 3.7 g (33%)of compound 26 as a dark red solid. ¹H NMR (DMSO-d6) δ 12.9 (br s, 1H),8.01 (d, J=8.7 Hz, 2H), 7.72 (d, J=8.7 Hz, 2H), 7.25 (s, 1H), 6.41 (s,1H), 6.24 (s, 2H), 3.83 (s, 3H), 3.76 (s, 3H),

4-[(E)-(2,5-Dimethoxy-4-{(E)-[4-(dimethylamino)phenyl]diazenyl}phenyl)diazenyl]benzoicAcid (27)

A solution of NaNO₂ (0.85 g, 12.3 mmol) in 8 mL of water was added viaan addition funnel over the course of 5 min to a cold (0-4° C.) stirredsuspension of compound 26 (3.7 g, 12.3 mmol) in a mixture of water (67mL) and conc. HCl (2.0 mL). The reaction was stirred at 0-4° C. for 30min and then at room temperature for 2 h before being used in the nextstep. A solution of N,N-dimethylaniline (2.4 mL) in 10 mL of MeOH wasadded in one portion to the diazonium salt from step 1 followed by 5.0 gof sodium acetate trihydrate. The resulting dark suspension was stirredat room temperature for 16 h and then filtered to collect theprecipitated solid. The crude dye was purified by trituration in hotMeOH (20 mL) followed by cooling and filtration to yield 2.2 g (41%) ofdye 27 as a black solid. ¹H NMR (DMSO-d6) δ 13.17 (br s, 1H), 8.11 (d,J=8.4 Hz, 2H), 7.93 (d, J=8.7 Hz, 2H), 7.80 (d, J=8.7 Hz, 2H), 7.39 (s,1H), 7.35 (s, 1H), 6.83 (d, J=9 Hz, 2H), 3.96 (s, 3H), 3.92 (s, 3H),3.06 (s, 6H).

Pentafluorophenyl4-[(E)-(2,5-dimethoxy-4-{(E)-[4-(dimethylamino)phenyl]diazenyl}phenyl)diazenyl]benzoate(28)

PFP-TFA (1.2 mL, 6.9 mmol) was added in two portions to a solution of 27(2.2 g, 5.07 mmol) and triethylamine (2 mL) in 50 mL of anhydrousCH₂Cl₂. After being stirred for 2 h the reaction was concentrated andthe obtained semi-solid residue resuspended in 2-propanol (25 mL). Theresulting solid was collected by filtration and dried in vacuo to affordcrude ester 28, which was then further purified by trituration in hot4:1 mixture of hexane and ethyl acetate (20 mL) followed by cooling andfiltration. Drying in vacuo afforded 2.2 g (72%) of PFP ester 28. ¹H NMR(CDCl₃) δ 8.34 (d, J=8.4 Hz, 2H), 8.05 (d, J=8.7 Hz, 2H), 7.96 (d, J=9Hz, 2H), 7.53 (s, 1H), 7.49 (s, 1H), 6.77 (d, J=9 Hz, 2H), 4.10 (s, 3H),4.07 (s, 3H), 3.13 (s, 6H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[4-[(E)-(2,5-dimethoxy-4-{(E)-[4-(dimethylamino)phenyl]diazenyl}phenyl)diazenyl]benzoyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (29)

A mixture of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (1 mmol), 28 (0.6 g, 1 mmol), DMF (5 mL) and triethylamine wasstirred at 50° C. for 20 h and then cooled to room temperature. Theresulting precipitate was collected by filtration, washed with DMF (2mL), CH₂Cl₂ (2×3 mL) and dried to yield 0.76 g (84%) of compound 29 as ablack solid (partial triethylammonium salt according to NMR analysis).¹H NMR (DMSO-d6) δ 11.80 (br s, 1H), 11.55 (br s, 1H), 8.22 (m, 2H),7.98 (m, 2H), 7.85 (m, 3H), 7.46 (s, 1H), 7.42 (s, 1H), 7.30 (d, J=8.7Hz, 2H), 7.10 (s, 1H), 6.89 (m, 3H), 4.65 (t, J=7.5 Hz, 2H), 4.21 (t,J=7.2 Hz, 2H), 4.02 (s, 3H), 3.99 (s, 3H), 3.39 (m, 4H), 3.11 (s, 6H),2.76 (m, 6H), 1.08 (t, J=7.2 Hz, 8H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[4-[(E)-(2,5-dimethoxy-4-{(E)-[4-(dimethylamino)phenyl]diazenyl}phenyl)diazenyl]benzoyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(30)

To a suspension of 29 (0.76 g, 0.84 mmol) in 10 mL of anhydrous DMF wasadded 0.5 mL of triethylamine and 0.2 mL (1.16 mmol) of PFP-TFA. Thereaction was stirred for 8 h and treated with another portion of PFP-TFA(0.15 mL) to complete the PFP ester formation. After being stirred for48 h the precipitated material was collected by filtration, washed withDMF (2×0.5 mL), acetone (4×5 mL) and dried in vacuo to give 0.44 (54%yield) of PFP ester 30 as a brown solid. ¹H NMR (DMSO-d6) δ 12.57 (br s,1H), 11.84 (br s, 1H), 8.44 (d, J=8.4 Hz, 1H), 8.22 (m, 1H), 8.00 (d,J=8.4 Hz, 2H), 7.86 (m, 4H), 7.62 (s, 1H), 7.45 (m, 4H), 7.16 (s, 1H),6.89 (d, J=9 Hz, 2H), 4.71 (t, J=7.5 Hz, 2H), 4.23 (t, J=7.2 Hz, 2H),4.03 (s, 3H), 4.00 (s, 3H), 3.55 (t, J=8.4 Hz, 2H), 3.35 (m, 2H), 3.13(s, 6H).

Ethyl5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylate(31)

To a suspension of ethyl 5-aminoindole-2-carboxylate (1.5 g, 7.34 mmol)in 40 mL of water was added 1.25 mL of conc. HCl. The stirred suspensionwas cooled in ice/water bath to 0-4° C. and treated with a solution ofNaNO₂ (0.55 g, 8 mmol) in 10 mL of water via an addition funnel over thecourse of 5 min. The reaction was stirred at 0-4° C. for 20 min thenwarmed to room temperature and stirred for another 30 min to give adark, mostly clear solution. 2-(Pyrrolidin-1-yl)-1,3-thiazole (C. Bogaet al., Org. Biomol. Chem. 2016, 14, 7061-7068) (1.5 mL, 9.7 mmol) wasadded in one portion followed by 2.7 g of sodium acetate trihydrate and7 mL of MeOH and stirring was continued for 3 h. The precipitated solidwas collected by filtration, washed with water and resuspended in 100 mLof MeOH. The suspension was heated to reflux then cooled and filtered tocollect the precipitated solid. Drying in vacuo afforded 2.35 g (86%) ofthe desired dye 31 as an orange solid. ¹H NMR (DMSO-d6) δ 12.14 (s, 1H),8.05 (s, 1H), 7.97 (d, J=1.5 Hz, 1H), 7.68 (dd, J₁=9 Hz, J₂=1.5 Hz, 1H),7.49 (d, J=9 Hz, 1H), 7.25 (s, 1H), 4.35 (q, J=7.9 Hz, 2H), 3.51(apparent br s, 4H), 2.00 (m, 4H), 1.34 (t, J=7.2 Hz, 3H).

5-{(E)-[2-(Pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylicAcid (32)

A suspension of 31 (2.35 g, 6.3 mmol) in a mixture of THF (135 mL), MeOH(90 mL) and 1N NaOH (45 mL) was heated at 50° C. with stirring for 2.5h. The reaction was cooled, neutralized with 45 mL of 1N HCl andconcentrated to about 80 mL. The precipitated solid was collected byfiltration, washed with water and dried to afford 2.17 g (100% yield) ofacid 32 as a brown solid. ¹H NMR (DMSO-d6) δ 13.08 (br s, 1H), 12.03 (s,1H), 8.06 (s, 1H), 7.97 (d, J=1.8 Hz, 1H), 7.68 (dd, J₁=9 Hz, J₂=1.8 Hz,1H), 7.47 (d, J=9 Hz, 1H), 7.20 (d, J=1.2 Hz, 1H), 3.52 (apparent br s,4H), 2.01 (m, 4H).

Pentafluorophenyl5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylate(33)

To a solution of 32 (1.0 g, 2.92 mmol) and triethylamine (2 mL) in 30 mLof anhydrous CH₂Cl₂ was added PFP-TFA in several 0.5 mL portions overthe course of 2 days until no more starting material was found by HPLCanalysis. A total of 1.5 mL (8.7 mmol) of PFP-TFA was added. Theprecipitated product was collected by filtration, washed with smallamount (approx. 2 mL) of CH₂Cl₂, 4:1 hexane-ethyl acetate and hexane.Drying in vacuo afforded 0.865 g (58%) of PFP ester 33 as a yellowsolid. ¹H NMR (DMSO-d6) δ 12.71 (d, J=1.2 Hz, 1H), 8.11 (s, 1H), 8.07(d, J=1.8 Hz, 1H), 7.80 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.71 (d, J=1.5 Hz,1H), 7.58 (d, J=9 Hz, 1H), 3.54 (apparent br s, 4H), 2.03 (m, 4H).

3,6,7,8-Tetrahydro-6-[(5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicacid (34)

To a solution of3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid, (0.79mmol) and 0.5 mL of triethylamine in 5 mL of DMF was added 0.4 g (0.79mmol) of PFP ester 33 and the reaction was stirred for 18 h.Precipitated material was collected by filtration, washed with MeOH (2×5ml) and dried in vacuo to give 0.414 g (99%) of compound 34 (partialtriethylammonium salt) as a yellow solid. ¹H NMR (DMSO-d6) δ 11.98 (s,1H), 11.73 (s, 1H), 8.26 (d, J=8.1 Hz, 1H), 8.06 (s, 1H), 7.99 (d, J=1.5Hz, 1H), 7.68 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.53 (d, J=9 Hz, 1H), 7.32(d, J=9 Hz, 1H), 7.26 (s, 1H), 7.00 (s, 1H), 4.65 (t, J=8.1 Hz, 2H),3.54 (apparent br s, 4H), 3.43 (t, J=8.1 Hz, 2H), 2.79 (q, J=7.2 Hz,2H), 2.03 (m, 4H), 1.08 (t, J=7.2 Hz, 3H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[(5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(35)

To a suspension of 34 (0.40 g, 0.76 mmol) in 9 mL of anhydrous DMF wasadded 0.5 mL of triethylamine and 0.2 mL (1.16 mmol) of PFP-TFA. Thereaction was stirred for 1 h and treated with another portion of PFP-TFA(0.2 mL) to complete the PFP ester formation. After being stirred for 2h the reaction was concentrated and the precipitated material wascollected by filtration. The solid was resuspended in 2-propanol (5 mL),washed with more 2-propanol (2×5 mL) and dried to yield 0.57 g (100%) ofPFP ester 35. According to NMR analysis the product was partially(approx. 40%) TFA-blocked at one of the indole NH-groups. ¹H NMR(DMSO-d6) δ 12.64 (s, 0.4H), 12.55 (s, 0.6H), 12.00 (s, 1H), 8.5-7.2(aromatic protons, 8H), 4.69 (m, 2H), 3.52 (m, 6H), 2.03 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (36)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.79 mmol) in 5 mL of anhydrous DMF was added 0.5 mL oftriethylamine followed by 0.4 g (0.79 mmol) of PFP ester 33. The mixturewas briefly stirred to initially give a clear solution which quicklyturned into a suspension due to the product precipitation. The reactionwas swirled for 18 h and the precipitated material collected byfiltration. The collected solid was washed with MeOH (3×5 mL), CH₂Cl₂(10 mL) and dried in vacuo to afford 0.59 g (100%, as triethylammoniumsalt) of compound 36 as an orange solid. ¹H NMR (DMSO-d6) δ 12.00 (s,1H), 11.78 (d, J=1.2 Hz, 1H), 11.63 (s, 1H), 8.27 (m, 2H), 8.07 (s, 1H),7.99 (d, J=1.5 Hz, 1H), 7.69 (dd, J₁=9 Hz, J₂=1.5 Hz, 1H), 7.54 (d, J=9Hz, 1H), 77.39 (d, J=9 Hz, 1H), 7.31 (d, J=9 Hz, 1H), 7.27 (s, 1H), 7.13(s, 1H), 6.94 (s, 1H), 4.67 (m, 4H), 3.53 (m, 4H), 3.42 (m, 4H), 2.72(q, J=7.2 Hz, 4H), 2.03 (m, 4H), 1.05 (t, J=7.2 Hz, 6H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(5-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(37)

To a suspension of 36 (0.58 g, 0.8 mmol) in DMF (9 mL) was addedtriethylamine (0.5 mL) followed by PFP-TFA (0.2 mL). The suspension wasstirred for 2 h and treated with another 0.2 mL portion of PFP-TFA andstirring was continued for 3 more days. The precipitated material wascollected by filtration, washed with 2-propanol (2×5 mL) and dried invacuo to afford 0.40 g (57%) of PFP ester 37 as a yellow solid. ¹H NMR(DMSO-d6) δ 12.56 (s, 1H), 11.99 (s, 1H), 11.82 (s, 1H), 8.42 (br d,J=8.7 Hz, 1H), 8.29 (br d, J=7.5 Hz, 1H), 8.07 (s, 1H), 8.00 (d, J=1.5Hz, 1H), 7.68 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.61 (d, J=1.2 Hz, 1H), 7.54(d, J=9 Hz, 1H), 7.44 (d, J=9 Hz, 1H), 7.39 (d, J=9 Hz, 1H), 7.27 (s,1H), 7.17 (s, 1H), 4.69 (m, 4H), 3.53 (m, 8H), 2.03 (m, 4H).

4-{(E)-[2,5-Dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1-methyl-1H-pyrrole-2-carboxylicAcid (38)

4-[[(1,1-dimethylethoxy)carbonyl]amino]-1-methyl-1H-pyrrole-2-carboxylicacid 90.49 g (2.04 mmol) was treated with 11 mL of conc. HCl for 30 min.The resultant solution was cooled to 0-4° C. and while being stirredtreated with a solution of sodium nitrite (0.15 g) in 3 mL of watermaintaining the temperature below 5° C. The cold reaction was stirred at0-4° C. for 2 h before being used in the next step. A solution of1-(2,5-dimethoxyphenyl)pyrrolidine (0.66 g, 3.2 mmol) in 50 mL of MeOHwas added with stirring to the solution of diazonium salt from step 1followed by 100 mL of 1 NaOH and the reaction was allowed to warm toroom temperature. After being stirred for 10 h the reaction wasconcentrated to approx. 100 mL and the resultant solid collected byfiltration. The solid was washed with water and dried in vacuo to afford0.275 g (37%) of dye 38 as a black solid. ¹H NMR (DMSO-d6) δ 7.65 (s,1H), 7.29 (s, 1H), 6.99 (d, J=1.8 Hz, 1H), 6.29 (s, 1H), 3.91 (s, 6H),3.74 (s, 3H), 3.36 (apparent br s, 4H), 1.90 (m, 4H).

Pentafluorophenyl4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1-methyl-1H-pyrrole-2-carboxylate(39)

To a solution of 38 (0.27 g, 0.75 mmol) and triethylamine (0.5 mL) in7.5 mL of anhydrous CH₂Cl₂ was added PFP-TFA in several 0.1 mL portionsover the course of 2 days until no more starting material was found byHPLC analysis. A total of 0.3 mL (1.7 mmol) of PFP-TFA was added. Thereaction was concentrated and the residue triturated with 2-propanol (5mL). The sold material was collected by filtration, washed with smallamount (approx. 2 mL) of MeOH and dried to afford 0.17 g (43%) of PFPester 39 as a brown orange solid. ¹H NMR (DMSO-d6) δ 7.99 (d, J=1.5 Hz,1H), 7.40 (d, J=1.8 Hz, 1H), 7.21 (s, 1H), 6.26 (s, 1H), 3.96 (s, 3H),3.89 (s, 3H), 3.71 (s, 3H), 3.49 (m, 4H), 1.88 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6[(4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1-methyl-1H-pyrrol-2-yI)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (40)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.33 mmol) in 2 mL of anhydrous DMF was added 0.3 mL oftriethylamine followed by 0.17 g (0.32 mmol) of PFP ester 39. Thereaction was stirred at 50° C. for 24 h and then concentrated. Theobtained dark residue was triturated with ethyl acetate (10 mL), theprecipitated material was collected by filtration, washed with MeOH (2×5mL) and dried to afford 0.203 g (84%) of compound 40 as a dark brownsolid. ¹H NMR (DMSO-d6) δ 13.00 (br s, 1H), 11.84 (s, 1H), 11.76 (s,1H), 8.28 (br d, J=7.5 Hz, 1H), 7.98 (br s, 1H), 7.64 (s, 1H), 7.34 (d,J=9H, 2H), 7.20 (s, 1H), 7.09 (s, 1H), 7.05 (s, 1H), 6.88 (s, 1H), 6.28(s, 1H), 4.65 (t, J=8.1 Hz, 2H), 4.44 (t, J=8.1 Hz, 2H), 3.88 (s, 3H),3.83 (s, 3H), 3.71 (s, 3H), 3.5-3.4 (m, 8H), 1.88 (m, 4H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6-[(4-{(E)-[2,5-dimethoxy-4-(pyrrolidin-1-yl)phenyl]diazenyl}-1-methyl-1H-pyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(41)

To a suspension of 40 (0.197 g, 0.27 mmol) in DMF (3 mL) was addedtriethylamine (0.2 mL) followed by PFP-TFA (0.1 mL). The suspension wasstirred for 20 h and treated with another 0.05 mL portion of PFP-TFA andstirring was continued for 1 more hour. DMF was removed and the residuetriturated with 2-propanol (5 mL). The resultant material was collectedby filtration, washed with 2-propanol (2×5 mL) and dried in vacuo toafford 0.218 g (90%) of PFP ester 41 as a brown solid. ¹H NMR (DMSO-d6)δ 12.54 (s, 1H), 11.78 (s, 1H), 8.40 (br d, J=7.8 Hz, 1H), 7.99 (br s,1H), 7.64 (s, 1H), 7.59 (s, 1H), 7.42 (d, J=9 Hz, 1H), 7.32 (d, J=9 Hz,1H), 7.20 (s, 1H), 7.11 (s, 1H), 6.88 (s, 1H), 6.28 (s, 1H), 4.66 (t,J=8.1 Hz, 2H), 4.44 (t, J=8.1 Hz, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.71(s, 3H), 3.5-3.4 (m, 8H), 1.88 (m, 4H).

Ethyl2-[(E)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yldiazenyl]-1,3-benzothiazole-6-carboxylate(42)

A solution of ethyl 2-amino-1,3-benzothiazole-6-carboxylate (2.22 g, 10mmol) in a 1:1 mixture of acetic and propionic acids (50 mL) was cooledto 0-4° C. and then treated with 3.1 mL of 40% nitrosylsulfuric acidover the course of 5 min. The resulting suspension was stirred at 0-4°C. for 5 min and then at room temperature for another 5 min to give aclear solution and then cooled back to 0-4° C. before being used in thenext step. A solution of julolidine (2.1 g, 12 mmol) in MeOH (15 mL) wascombined with a solution of sulfamic acid (0.3 g) in water (100 mL) andthen cooled to 0-4° C. To this mixture was added the previously prepareddiazonium salt over the course of 5 min. The reaction was stirred for 1h and then diluted with water (400 mL) and neutralized with 90 mL oftriethylamine. The precipitated solid was extracted with ethyl acetate,washed with saturated NaHCO₃, dried over Na₂SO₄ and concentrated underreduced pressure. The obtained residue was triturated with warm 1:1hexane-ethyl acetate (30 mL) and the resultant solid collected byfiltration. Drying under reduced pressure afforded 0.8 g (48%) ofsufficiently pure dye 42 as a dark purple solid. ¹H NMR (CDCl₃) δ 8.51(d, J=1.5 Hz, 1H), 8.11 (skewed dd, J₁=8.7 Hz, J₂=1.5 Hz, 1H), 8.01(skewed d, J=8.7 Hz, 1H), 7.61 (s, 2H), 4.42 (q, J=7.2 Hz, 2H), 3.39 (t,J=6 Hz, 4H), 2.80 (t, J=6 Hz, 4H), 2.00 (m, 4H), 1.43 (t, J=7.2 Hz, 3H).

2-[(E)-2,3,6,7-Tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yldiazenyl]-1,3-benzothiazole-6-carboxylicAcid (43)

To a solution of 42 (0.35 g, 0.86 mmol) in a mixture of THF (18 mL) andMeOH (12 mL) was added 6 mL of 1N NaOH. The reaction was stirred at 50°C. for 1.5 h then cooled, neutralized with 1N HCl (6 mL) andconcentrated under reduced pressure to about 10 mL. The resultingprecipitate was collected by filtration, washed with water and dried toafford 0.32 g (98%) of compound 43 as a black solid. ¹H NMR (DMSO-d6) δ13.0 (br s, 1H), 8.54 (d, J=1.5 Hz, 1H), 7.99 (skewed dd, J₁=8.7 Hz,J₂=1.5 Hz, 1H), 7.93 (skewed d, J=8.7 Hz, 1H), 7.47 (s, 2H), 3.43 (t,J=6 Hz, 4H), 2.77 (t, J=6 Hz, 4H), 1.90 (m, 4H).

Pentafluorophenyl2-[(E)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yldiazenyl]-1,3-benzothiazole-6-carboxylate(44)

To a solution of 43 (0.32 g, 0.84 mmol) and triethylamine (0.5 mL) in 10mL of anhydrous DMF was added PFP-TFA in several portions over thecourse of 3 hours until no more starting material was found by HPLCanalysis. A total of 0.5 mL (2.9 mmol) of PFP-TFA was added. Thereaction was concentrated under reduced pressure and the residuechromatographed on silica eluting with a gradient of ethyl acetate inhexane. Concentration of the pure product fractions afforded 0.13 g(28%) of PFP ester 44 as a dark purple solid. ¹H NMR (DMSO-d6) δ 8.86(d, J=1.8 Hz, 1H), 8.18 (skewed dd, J₁=8.4 Hz, J₂=1.8 Hz, 1H), 8.05(skewed d, J=8.4 Hz, 1H), 7.52 (s, 2H), 3.47 (t, J=6 Hz, 4H), 2.79 (t,J=6 Hz, 4H), 1.92 (m, 4H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yldiazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (45)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.24 mmol) in 2 mL of anhydrous DMF was added 0.2 mL oftriethylamine followed by 0.13 g (0.24 mmol) of PFP ester 44. Afterbeing stirred at 50° C. for 6 h the reaction was concentrated underreduced pressure to yield a dark purple residue. To isolate the productthe residue was resuspended in MeOH (10 mL), the precipitated materialwas collected by filtration, washed with MeOH (2×5 mL) and dried toafford 0.181 g (100%) of compound 45 as a dark purple solid. ¹H NMR(DMSO-d6) δ 12.9 (br s, 1H), 11.84 (s, 1H), 11.76 (s, 1H), 8.4-8.1 (m,3H), 7.98 (d, J=8.4 Hz, 1H), 7.68 (br d, J=7.8 Hz, 1H), 7.48 (s, 2H),7.3 (m, 2H), 7.07 (d, J=1.5 Hz, 1H), 7.05 (d, J=1.5 Hz, 1H), 4.63 (t,J=7.8 Hz, 2H), 4.20 (t, J=7.5 Hz, 2H), 3.4 (m, 6H), 3.27 (m, 2H), 2.78(t, J=6 Hz, 4H), 1.91 (m, 4H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yldiazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (46)

To a suspension of 45 (0.175 g, 0.23 mmol) in DMF (2.5 mL) was addedtriethylamine (0.2 mL) followed by two 0.1 mL portions of PFP-TFA overthe course of 3 hours. DMF was removed under reduces pressure and theresidue triturated with 2-propanol (5 mL). The resultant insolublematerial was collected by filtration, washed with 2-propanol (2×5 mL)and dried under reduced pressure to afford 0.20 g (95%) of PFP ester 46as a dark purple solid. ¹H NMR (DMSO-d6) δ 12.53 (s, 1H), 11.78 (s, 1H),8.6-7.0 (aromatic protons, 11H), 4.66 (t, J=7.8 Hz, 2H), 4.20 (t, J=7.5Hz, 2H), 3.9-3.0 (m, 8H), 3.27 (m, 2H), 2.77 (m, 4H), 1.90 (m, 4H).

Ethyl6-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazole-2-carboxylate(47)

To a suspension of ethyl 5-amino-1,3-benzothiazole-2-carboxylate(prepared by Fe/NH₄Cl/water/EtOH reduction of ethyl5-nitro-1,3-benzothiazole-2-carboxylate (U.S. Pat. No. 8,586,759)) (0.9g, 3.57 mmol) in water (20 mL) was added conc. HCl (0.625 mL). Themixture was stirred at room temperature for 5 min and then cooled to0-4° C. A solution of NaNO₂ (0.246 g) in water (5 mL) was added withstirring over the course of 5 min. The reaction was stirred for 10 minand then warmed to room temperature and stirred for another 10 min. Tothe resultant solution of diazonium salt was added a solution of2-(pyrrolidin-1-yl)-1,3-thiazole (0.66 g, 4.28 mmol) in 10 mL ofmethanol followed by solid sodium acetate, trihydrate (1.35 g). Thereaction was stirred at room temperature for 2 h to give a thick orangesuspension. The precipitated material was collected by filtration,washed with water and dried to give crude product, which was thenre-crystallized from ethyl acetate to afford 0.91 g (66%) of dye 47 asan orange solid. ¹H NMR (DMSO-d6) δ 8.45 (d, J=1.8 Hz, 1H), 8.26 (s,1H), 8.24 (d, J=9 Hz, 1H), 7.92 (dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 4.44 (q,J=6.9 Hz, 2H), 3.56 (br s, 4H), 2.14 (m, 4H), 1.34 (t, J=6.9 Hz, 3H).

6-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazole-2-carboxylicAcid (48)

To a solution of 47 (0.90 g, 2.32 mmol) in a mixture of THF (48 mL) andMeOH (32 mL) was added 16 mL of 1N NaOH. The reaction was stirred at 50°C. for 1.5 h then cooled, neutralized with 1N HCl (16 mL) andconcentrated under reduced pressure to about 30 mL. The resultingprecipitate was collected by filtration, washed with water and dried toafford 0.81 g (97%) of compound 48 as a black solid. ¹H NMR (DMSO-d6) δ8.40 (s, 1H), 8.22 (s, 1H), 8.19 (d, J=8.4 Hz, 1H), 7.88 (d, J=8.4 Hz,1H), 3.55 (br s, 4H), 2.01 (br s, 4H).

Pentafluorophenyl6-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazole-2-carboxylate(49)

Prior to reaction starting acid 48 (0.8 g, 2.22 mmol) was dried byco-evaporation with anhydrous DMF (10 mL) supplemented with 0.5 mL oftriethylamine and then re-dissolved in a fresh portion of DMF (10 mL).To this solution was added triethylamine (0.5 mL) followed by PFP-TFA(0.4 mL, 2.3 mmol). Within minutes a thick suspension was obtained. Thereaction was agitated on an orbital shaker for 1 h, then concentratedunder reduced pressure and the resultant residue chromatographed onsilica eluting with ethyl acetate. Concentration of theproduct-containing fractions and re-crystallization from ethyl acetateafforded 0.803 g (69%) of PFP ester 49 as a red solid. ¹H NMR (CDCl₃) δ8.34 (d, J=1.8 Hz, 1H), 8.32 (d, J=9 Hz, 1H), 8.14 (s, 1H), 8.07 (dd,J₁=9 Hz, J₂=1.8 Hz, 1H), 3.67 (br s, 4H), 2.03 (m, 4H).

3,6,7,8-Tetrahydro-6-[(6-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (50)

To a solution of3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid (0.69mmol) and 0.45 mL of triethylamine in 4.5 mL of DMF was added 0.35 g(0.67 mmol) of PFP ester 49. The reaction was stirred for 2 hours at 50°C. and cooled to room temperature. The precipitated material wascollected by filtration, washed with DMF (2 mL), acetone (3×15 mL),methanol (2×10 ml) and dried to give 0.375 g (100%) of compound 50 as abrick-red solid. ¹H NMR (DMSO-d6) δ 11.70 (s, 1H), 8.39 (s, 1H), 8.28(d, J=9.3 Hz, 1H), 8.22 (s, 1H), 8.17 (d, J=9 Hz, 1H), 7.87 (d, J=9 Hz,1H), 7.32 (d, J=9.3 Hz, 1H), 6.93 (s, 1H), 4.89 (m, 2H), 3.7-3.3 (m,6H), 2.01 (br s, 4H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[(6-{(E)-[2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(51)

PFP-TFA (a total of 0.35 mL, 1.63 mmol) was added in several portionsover the course of 20 h to a suspension of 50 (0.36 g, 0.66 mmol) andtriethylamine (0.4 mL) in 5 mL of anhydrous DMF until no more startingmaterial was found by HPLC analysis. The reaction was concentrated underreduced pressure and the obtained semi-solid residue resuspended inmethanol (25 mL). The resulting solid was collected by centrifugation at4,000 rpm for 10 min, methanol decanted and the procedure was repeatedtwo more times. Drying under reduced pressure afforded 0.340 g (72%) ofPFP ester 51 as a brick-red solid. ¹H NMR (DMSO-d6) δ 12.62 (s, 1H),8.48 (8.46 (d, J=9 Hz, 1H), 8.44 (d, J=1.5 Hz, 1H), 8.25 (s, 1H), 8.21(d, J=9 Hz, 1H), 7.90 (dd, J₁=9 Hz, J₂=1.5 Hz, 1H), 7.62 (s, 1H), 7.46(d, J=9 Hz, 1H), 4.95 (t, J=8.1 Hz, 2H), 3.55 (br s, 4H), 3.50 (t, J=8.4Hz, 2H), 2.03 (m, 4H).

4-Phenyl-2-(pyrrolidin-1-yl)-1,3-thiazole (52)

A mixture of 2-Bromo-4-phenyl-1,3-thiazole (5.4 g, 22.5 mmol) andpyrrolidine (5 mL, 60 mmol) was heated at 80° C. for 2.5 h, then cooledand concentrated. The resultant semi-solid was resuspended in 1:2ethyl-acetate-hexane and the precipitate pyrrolidinium bromide removedby filtration. The filtrate was concentrated and the residuechromatographed on silica eluting with 1:2 ethyl acetate-hexane toafford 5.15 g of compound 52 as a colorless crystalline solid. ¹H NMR(CDCl₃) δ 7.86 (m, 1H), 7.83 (m, 1H), 7.36 (dt, J₁=6 Hz, J₂=1.5 Hz, 2H),7.26 (skewed dt, J₁=6 Hz, J₂=1.5 Hz, 1H), 6.66 (s, 1H), 3.51 (m, 4H),2.04 (m, 4H).

Ethyl5-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylate(53)

To a suspension of ethyl 5-aminoindole-2-carboxylate (0.75 g, 3.67 mmol)in 20 mL of water was added 0.625 mL of conc. HCl. The stirredsuspension was cooled in ice/water bath to 0-4° C. and treated with asolution of NaNO₂ (0.275 g, 3.98 mmol) in 5 mL of water over the courseof 5 min. The reaction was stirred at 0-4° C. for 30 min then warmed toroom temperature and stirred for another 30 min. To this solution wasadded a solution of 52 (1.1 g, 4.85 mmol) in 16 mL of methanol followedby sodium acetate, trihydrate (1.35 g) and stirring was continued for 5h. The resultant precipitated was collected by filtration, washed withwater, resuspended in 20 mL of MeOH and dried. The crude dye wasre-crystallized from methanol to afford 1.42 g (87%) of dye 53 as anorange-red solid. ¹H NMR (CDCl₃) δ 8.96 (br s, 1H), 8.34 (dd, J₁=8.1 Hz,J₂=1.5 Hz, 2H), 8.07 (d, J=1.8 Hz, 1H), 7.85 (dd, J₁=9 Hz, J₂=1.8 Hz,1H), 7.55-7.35 (m, 4H), 7.30 (s, 1H), 4.42 (q, J=6.9 Hz, 2H), 3.66 (brs, 4H), 2.10 (m, 4H), 1.43 (t, J=6.9 Hz, 3H).

5-{(E)-[4-Phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylicAcid (54)

A suspension of 53 (1.3 g, 2.92 mmol) in a mixture of THF (60 mL) andmethanol (40 mL) was added 1N NaOH (20 mL). The reaction was stirred at50° C. for 1.5 h, then cooled, neutralized with 1N HCl (20 mL) andconcentrated to approx. 40 mL. The precipitated material was collectedby filtration, washed with water and dried to afford 1.22 g (100%) ofacid 54 as a dark brown solid. ¹H NMR (DMSO-d6) δ 12.03 (s, 1H), 8.29(d, J=8.7 Hz, 2H), 7.98 (d, J=1.5 Hz, 1H), 7.69 (dd, J₁=9 Hz, J₂=1.8 Hz,1H), 7.6-7.4 (m, 4H), 7.20 (d, J=1.5 Hz, 1H), 3.59 (br s, 4H), 2.04 (brs, 4H).

Pentafluorophenyl5-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indole-2-carboxylate(55)

Acid 54 (1.22 g, 2.92 mmol) was first dried by co-evaporation withanhydrous DMF (15 mL) then re-dissolved in fresh portion of DMF (15 mL)and treated with triethylamine (1 mL) and PFP-TFA (0.57 mL, 3.3 mmol).After being stirred at room temperature for 3 h the reaction wasconcentrated under reduced pressure and the resultant sold resuspendedin methanol (15 mL). The insoluble material was collected by filtration,washed with methanol (2×5 mL) and dried to afford 1.71 g (87% as a DMFadduct) of PFP ester 55 as an orange solid. ¹H NMR (CDCl₃) δ 9.09 (br s,1H), 8.34 (m, 2H), 8.11 (d, J=1.8 Hz, 1H), 8.02 (br s, 1H, DMF), 7.94(dd, J₁=9 Hz, J₂=1.8 Hz, 1H), 7.62 (d, J=1.8 Hz, 1H), 7.55-7.35 (m, 4H),3.67 (br s, 4H), 2.96 (s, 3H, DMF), 2.89 (s, 3H, DMF), 2.12 (m, 4H).

3,6,7,8-Tetrahydro-6-[(5-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (56)

To a solution of3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid (0.79mmol) in 5 mL of DMF was added 0.5 mL of triethylamine followed by 0.53g (0.79 mmol) of PFP ester 55. The mixture was stirred for a few minutesto give an almost complete solution before product precipitation began.The suspension was heated at 50° C. for 2 h and then diluted withmethanol (10 mL). The precipitate was collected by filtration, washedwith methanol (3×10 mL) and dried to afford 0.545 g (100% astriethylammonium salt) of compound 56 as an orange-red solid. ¹H NMR(DMSO-d6) δ 11.95 (s, 1H), 11.56 (s, 1H), 8.31 (m, 2H), 8.22 (br d,J=8.4 Hz, 1H), 7.99 (d, J=1.5 Hz, 1H), 7.69 (dd, J₁=9 Hz, J₂=1.8 Hz,1H), 7.6-7.4 (m, 4H), 7.29 (d, J=9 Hz, 1H), 7.26 (s, 1H), 6.89 (s, 1H),4.65 (t, J=8.1 Hz, 2H), 3.60 (br s, 4H), 3.41 (t, J=8.4 Hz, 2H), 2.67(q, J=6.9 Hz, 6H), 2.04 (m, 4H), 1.03 (t, J=7.2 Hz, 9H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[(5-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1H-indol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(57)

Before starting the reaction, acid 56 (0.545 g, 0.79 mmol) was dried byco-evaporation with anhydrous DMF (20 mL), then re-dissolved in freshportion of DMF (6 mL) and treated with triethylamine (0.5 mL). PFP-TFAwas added in several 0.1 mL portions over the period of 9 h until nomore starting acid 56 was found by HPLC analysis. A total of 0.8 mL(4.66 mmol) of PFP-TFA was added. The resultant suspension wasconcentrated and the residue triturated in methanol (10 mL). The solidwas collected by filtration, washed with methanol and dried to afford0.6 g (99%) of PFP ester 57 as a red-orange solid. This product waspartially TFA-blocked at the indole NH groups. ¹H NMR (DMSO-d6) δ 12.55(s, 0.4H), 11.98 (s, 1H), 8.33 (m, 2H), 8.00 (s, 1H), 7.8-7.2 (aromaticprotons, approx. 9H), 4.70 (t, J=8.4 Hz, 2H), 3.60 (br s, 4H), 5.52 (mobscured by water signal, 2H), 2.05 (br s, 4H).

3,6,7,8-Tetrahydro-6-[[6-acetyl-3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (58)

Acetic anhydride (0.12 mL, 1.27 mmol) was added to a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (1.0 mmol) and 0.4 mL of triethylamine in 10 mL of DMF.Precipitation of the product started within minutes after the addition.Reaction was allowed to continue for 15 h, the precipitated material wascollected by filtration, washed with acetone (3×15 mL) and dried toafforded 0.36 g (84%) of 58 as an off-white solid. ¹H NMR (DMSO-d6) δ13.0 (br s, 1H), 11.85 (d, J=1.8 Hz, 1H), 11.70 (d, J=1.8 Hz), 8.28 (brd, J=8.7 Hz, 1H), 8.17 (d, J=8.7 Hz, 1H), 7.33 (d, J=9.3 Hz, 1H), 7.29(d, J=8.4 Hz, 1H), 7.07 (d, J=1.2 Hz, 1H), 7.05 (d, J=1.5 Hz, 1H), 4.65(t, J=8.1 Hz, 2H), 4.22 (t, J=8.4 Hz, 2H), 3.43 (t, 8.1 Hz, 2H), 3.39 (mpartially obscured by the water signal, 2H), 2.18 (s, 3H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[6-acetyl-3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(59)

PFP-TFA was added in several portions (0.04-0.12 mL each) with stirringover the period of 20 h to a solution of 58 (0.35 g, 0.82 mmol) andtriethylamine (0.4 mL) in 10 mL of anhydrous DMF until no more startingmaterial was found by HPLC analysis. DMF was removed under reducedpressure and the residue resuspended in acetone (10 mL). The insolublematerial was collected by filtration, washed with acetone and dried toafford 0.46 g (94%) of 59 as an off-white solid. ¹H NMR (DMSO-d6) δ12.56 (d, J=1.8 Hz, 1H), 11.73 (d, J=1.5 Hz), 8.44 (br d, J=9.3 Hz, 1H),8.19 (d, J=9 Hz, 1H), 7.97 (s, 1H), 7.61 (d, J=1.5 Hz, 1H), 7.45 (d, J=9Hz, 1H), 7.31 (d, 9 Hz, 1H), 7.08 (d, J=1.5 Hz, 1H), 4.69 (t, J=8.4 Hz,2H), 4.22 (t, J=8.4 Hz, 2H), 3.49 (t, 8.4 Hz, 2H), 3.39 (m partiallyobscured by the water signal, 2H), 2.19 (s, 3H).

Example 2. Preparation of DSQ-Modified 6-aminohexane-1,2-diol SynthesisSolid Supports(6-N-(Fluorenylmethoxycarbonyl)amino-1-(4,4′-dimethoxytriphenylmethoxy)hex-2-yl)oxy-4-oxobutanoylPolystyrene Support (ID#349 PS)

(6-N-(Fluorenylmethoxycarbonyl)amino-1-(4,4′-dimethoxytriphenylmethoxy)hex-2-yl)oxy-4-oxobutanoylPolystyrene Support (ID #349 PS) (DMT loading 20 μmol/g) was prepared inthe same manner as the CPG support described in U.S. Pat. No. 6,492,346starting from aminomethyl polystyrene support (33 mmol/g, AppliedBiosystems PN 360865C).

Example 3. Preparation of Polystyrene Supports General Procedure for thePreparation of Polystyrene Supports ID#473, 474-477, 479, 481-488

Step 1. FMOC Deprotection

Polystyrene support (ID#349 PS) (2.0 g) was resuspended in 15 mL of 0.2M solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF andagitated on an orbit shaker for 10 min. DMF was removed by filtrationand the procedure was repeated two more times. The polystyrene supportwas then washed with either DMF or N-Methyl-2-pyrrolidone (NMP) (3×50mL), depending on the choice of solvent in the next step, andimmediately reacted with a PFP ester as described below.

Step 2. PFP Ester Coupling

A solution of one of the PFP ester (50 μmol) synthesized as describedabove in Example 1 (complete list of PFP esters used in this reaction isshown in Table 3) and triethylamine (0.2 mL) in 8.5 mL of a solventindicated in Table 3 was combined with the polystyrene support fromStep 1. The suspension was agitated for the period of time indicated inTable 3 then filtered, washed with the same solvent (3×50 mL) andimmediately taken to the next step.

TABLE 3 Step 2 Resultant Step 2 reaction Polystyrene PFP ester time (h)Solvent Support ID #  5 18 DMF 473 10 18 NMP 475 20 18 NMP 476 15 18 NMP477 25 12 NMP 479 30 16 NMP 481 59 20 NMP 482 41 12 DMF 483 35 20 NMP484 37 24 NMP 485 46 20 DMF 486 51 22 NMP 487 49 24 NMP 488 33 24 NMP489 57 15 NMP 490

Step 3. Blocking Unreacted Amino Groups

The support from step 2 was resuspended in 18 mL of pyridine and treatedwith 2 mL of acetic anhydride. The suspension was agitated for 30 min,then filtered, washed with DMF (3×50 mL) and acetone (3×50 mL) and driedunder reduced pressure.

Example 4. Preparation of Polystyrene Support ID #478 Ethyl2-[(E)-{4-[(6-acetyloxyhexyl)(methyl)amino]phenyl}diazenyl]-1,3-benzothiazole-6-carboxylate(60)

To a cold (0-4° C.) suspension of ethyl2-amino-1,3-benzothiazole-6-carboxylate (0.444 g, 2.0 mmol) in a mixtureof acetic (5 mL) and propionic (5 mL) acids was added dropwise 0.62 mLof 40% nitrosylsulfuric acid with stirring. After being stirred for 3 han almost clear yellow-orange solution was obtained which wasimmediately used in the next step. The solution of diazonium salt wasadded in several portions to a cold (0-4° C.) mixture prepared bycombining a solution of 6-[methyl(phenyl)amino]hexyl acetate (WO2008008481) (0.6 g, 2.4 mmol) in 2.5 mL of MeOH and a solution ofsulfamic acid (0.060 g) in 20 mL of water. The reaction was stirred at0-4° C. for 2 hours, neutralized by adding saturated NaHCO₃ in severalportions and the precipitated material extracted with ethyl acetate. Theextract was concentrated under reduced pressure and the obtained residuetriturated with 4:1 hexane-ethyl acetate (30 mL). The resulting solidwas collected by filtration, washed with MeOH (15 mL) and dried toafford 0.415 g (42%) of azo-dye 60 as a black solid. ¹H NMR (CDCl₃) δ8.55 (s, 1H), 8.13 (skewed dd, J₁=8.4 Hz, J₂=1.2 Hz, 1H), 8.07 (skewedd, J=8.4 Hz, 1H), 8.00 (d, J=9.3 Hz, 2H), 6.76 (d, J=9.3 Hz, 2H), 4.43(q, J=7.2 Hz, 2H), 4.07 (t, J=6.6 Hz, 2H), 3.49 (t, J=7.5 Hz, 2H), 3.14(s, 3H), 2.05 (s, 3H), 1.66 (m, 4H), 1.44 (m, 7H).

2-[(E)-{4-[(6-hydroxyhexyl)(methyl)amino]phenyl}diazenyl]-1,3-benzothiazole-6-carboxylicAcid (61)

1N NaOH (6 mL) was added to a solution of azo-dye 60 (0.415 g, 0.86mmol) in a mixture of THF (18 mL) and MeOH (12 mL). The reaction washeated at 50° C. with stirring for 1.5 h, then cooled to roomtemperature, neutralized with 1 N HCl (6 mL) and concentrated underreduced pressure to about 10 mL. The resulting precipitate was collectedby filtration, washed with water and dried to yield 0.356 g (100%) ofcompound 61 as a black solid. ¹H NMR (DMSO-d6) δ 13.08 (s, 1H), 8.58 (s,1H), 7.99 (s, 2H), 7.83 (d, J=9.3 Hz, 2H), 6.92 (d, J=9.3 Hz, 2H), 4.32(br s, 1H), 3.51 (t, J=6.9 Hz, 2H), 3.35 (m, 2H), 3.12 (s, 3H), 1.56 (m,2H), 1.36 (m, 2H), 1.29 (m, 4H).

Pentafluorophenyl2-[(E)-{4-[(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhexyl)(methyl)amino]phenyl}diazenyl]-1,3-benzothiazole-6-carboxylate(62)

Compound 61 (0.355 g, 0.86 mmol) was dried by co-evaporation withanhydrous pyridine (25 mL) and then re-dissolved in a fresh portion ofpyridine (10 mL). Dimethoxytrityl chloride (0.336 g, 1.0 mmol) wasadded, the reaction was stirred for 3 h and treated with PFP-TFA (0.4mL, 2.3 mmol) and allowed to react for another 3 h. Pyridine was removedunder reduced pressure and the residue partitioned between ethyl acetateand 10% citric acid. The organic phase was dried over Na₂SO₄,concentrated and the resulting material chromatographed on silicaeluting with a gradient of ethyl acetate in hexane (30 to 50%).Fractions containing the desired product were concentrated to give 0.45g (59%) of PFP ester 62 as an amorphous dark purple solid. This materialwas sufficiently pure for subsequent use.

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-{4-[(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhexyl)(methyl)amino]phenyl}diazenyl]-1,3-benzothiazol-6-yI)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (63)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (0.45 mmol) in 8 mL of anhydrous DMF was added 0.5 mL oftriethylamine followed by 0.4 g (0.45 mmol) of PFP ester 62. After beingstirred at room temperature for 20 h the reaction was concentrated underreduced pressure. The obtained residue was resuspended in acetone (10mL), the precipitated material was collected by filtration, washed withmore acetone (2×5 mL) and dried to afford 0.41 g (84%) of compound 63 asa black solid. ¹H NMR (DMSO-d6) δ 11.85 (s, 1H), 11.78 (s, 1H), 8.5-6.9(aromatic protons, 26H), 4.64 (m, 2H), 4.21 (m, 2H), 3.74 (s, 6H),3.6-3.2 (m, 8H), 3.13 (s, 3H), 1.58 (m, 4H), 1.36 (m, 2H), 1.28 (m, 2H).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-{4-[(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhexyl)(methyl)amino]phenyl}diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(64)

To a suspension of 63 (0.285 g, 0.26 mmol) in DMF (4 mL) was addedtriethylamine (0.2 mL) followed PFP-TFA (0.1 mL). After being stirredfor 1 h the reaction was concentrated and the resulting residueresuspended in acetone (10 mL). The solid material was collected byfiltration, washed with acetone (2×5 mL) and dried under reducedpressure to afford 0.25 g (77%) of PFP ester 64 as a dark brawn solid.¹H NMR (DMSO-d6) δ 12.54 (s, 1H), 11.80 (s, 1H), 8.5-6.9 (aromaticprotons, 26H), 4.68 (m, 2H), 4.22 (m, 2H), 3.74 (s, 6H), 3.6-3.2 (m,8H), 3.13 (s, 3H), 1.58 (m, 4H), 1.36 (m, 2H), 1.28 (m, 2H).

Preparation of Polystyrene Support ID #478

(N-MMT-5-aminopentyl)glycolate polystyrene support (U.S. Pat. No.7,381,818) (5.0 g, 20 umol/g MMT loading) was deprotected by suspendingin 30 mL of 3% trichloactetic acid in CH₂Cl₂ and filtration. Theprocedure was repeated approx. 10 times until no more yellow color wasobserved. The support then was washed with CH₂Cl₂, 10%triethylamine/CH₂Cl₂ and N-methylpyrrolidinone (NMP) before beingresuspended in a solution of 64 (0.156 g, 0.12 mmol, 25 umol/g offering)and triethylamine (0.2 mL) in 25 mL of NMP. The suspension was swirledon an orbital shaker for 18 h then filtered and washed with NMP. Toblock unreacted aminogroups the support was suspended in pyridine (22.5mL), treated with acetic anhydride (2.5 mL) and swirled for 30 min. Theblocking reagents were removed by filtration and the support was washedwith several 50 mL portions of NMP and acetone followed by drying underreduced pressure to afford polystyrene support ID #478 as a light purplesolid. DMT loading was 19.0 mol/g according to a colorimetric acidcleavage test.

Example 5. Preparation of Polystyrene Support ID #4803-[Methyl(phenyl)amino]propyl Acetate (65)

A mixture of N-methylaniline (23.6 mL, 0.218 mol), 3-chloropropylacetate(32.4 ml, 0.266 mol), diisopropylethylamine (46.4 mL, 0.267 mol) andsodium iodide (2.2 g, 14.7 mmol) was heated at 120° C. with stirring for7 h before being cooled and partioned between water (500 mL) and ethylacetate (200 mL). The organic phase was washed with brine, dried overNa₂SO₄ and concentrated. The resultant crude product was purified byvacuum distillation at 1 mmHg with the desired product distilling atapprox. 110° C. yielding 35.8 g (80%) of 3-[methyl(phenyl)amino]propylacetate (65) as a pale yellow liquid. ¹H NMR (CDCl₃) δ 7.7 (m, 2H), 6.7(m, 3H), 4.11 (t, J=6.3 Hz, 2H), 3.42 (t, J=6.9 Hz, 2H), 2.93 (s, 3H),2.07 (s, 3H), 1.91 (p, J=6.6 Hz, 2H).

2-[(E)-(4-{[3-(Acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazole-6-carboxylicAcid (66)

To a cold (0-4° C.) suspension of 2-amino-1,3-benzothiazole-6-carboxylicacid (1.25 g, 6.25 mmol) in a mixture of acetic (16 mL) and propionic(16 mL) acids was added dropwise 40% nitrosylsulfuric acid (2.0 mL) overthe course of 5 min. The reaction was stirred at 0-4° C. for 2 h andthen at room temperature for 2 h to give a light yellow suspension. Thissuspension was added slowly (approx. 5 min) to a cold (0-4° C.) mixtureprepared by combining a solution of 3-[methyl(phenyl)amino]propylacetate (65) (1.6 g, 7.7 mmol) in methanol (8 mL) and a solution ofsulfamic acid (0.2 g) in water (65 mL). After being stirred for 1 h, thereaction was neutralized by adding 65 mL of triethylamine and dilutedwith water (500 mL). The precipitated solid was collected by filtration,washed with water and dried. The crude dye was triturated with 4:1hexane-ethyl acetate (20 mL) and the solid was collected by filtration.Drying under reduced pressure afforded 1.66 g (64%) of sufficiently pureazo-dye 66 as a black solid. ¹H NMR (DMSO-d6) δ 8.62 (s, 1H), 8.04 (s,2H), 7.89 (d, J=9 HZ, 2H), 6.98 (d, J=9 Hz, 2H), 4.07 (t, J=6 Hz, 2H),3.66 (t, J=6.6 Hz, 2H), 3.16 (s, 3H), 2.04 (s, 3H), 1.93 (p, J=6.3 Hz,2H).

Pentafluorophenyl2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazole-6-carboxylate(67)

To a solution of 66 (1.66 g, 4.0 mmol) and triethylamine (0.5 mL) in 10mL of anhydrous CH₂Cl₂ was added PFP-TFA in two portions over the courseof 3 hours until no more starting material was found by HPLC analysis. Atotal of 1.1 mL (6.4 mmol) of PFP-TFA was added. The reaction wasconcentrated under reduced pressure and the residue resuspended in 20 mLof methanol. The precipitate was collected by filtration, washed withmethanol and dried to afford 1.88 g (81%) of PFP ester 67 as dark purplesolid. ¹H NMR (CDCl₃) δ 8.72 (s, 1H), 8.27 (skewed dd, J₁=8.4 Hz, J₂=1.2Hz, 1H), 8.16 (d, J=8.7 Hz, 1H), 8.03 (d, J=9 Hz, 2H), 6.79 (d, J=9 Hz,2H), 4.16 (t, J=6 Hz, 2H), 3.63 (t, J=7.2 Hz, 2H), 3.18 (s, 3H), 2.11(s, 3H), 2.03 (m, 2H).

3,6,7,8-Tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicacid (68)

To a solution of3,6,7,8-tetrahydro-6-[(3,6,7,8-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-2-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole]-2-carboxylicacid (3.05 mmol) in 20 mL of anhydrous DMF was added 2 mL oftriethylamine followed by 1.8 g (3.11 mmol) of PFP ester 67. Thereaction was stirred at 50° C. for 3 h and then concentrated. Theobtained dark residue was triturated with acetone (30 mL), the resultantsolid was collected by filtration, washed with acetone (2×10 mL) anddried to afford 2.55 g (107% due to a partial triethylammonium saltform) of compound 68 as a dark brown-purple solid. ¹H NMR (DMSO-d6) δ11.77 (s, 1H), 11.62 (s, 1H), 8.32 (s, 1H), 8.2 (m, 2H), 8.07 (d, J=8.4Hz, 1H), 7.89 (d, J=9 Hz, 2H), 7.74 (br d, J=8.1 Hz, 1H), 7.35 (br s,1H), 7.29 (d, J=9 Hz, 1H), 7.08 (s, 1H), 6.98 (d, J=9.3 Hz, 2H), 6.92(s, 1H), 4.63 (t, J=7.5 Hz, 2H), 4.21 (t, 2H), 4.07 (t, J=6 Hz, 2H),3.66 (t, J=6.3 Hz, 2H), 3.40 (t, J=7.5 Hz, 2H), 3.31 (t, J=7.5 Hz, 2H),3.16 (s, 3H), 2.73 (m, 5H, Et₃NH⁺), 2.04 (s, 3H), 1.95 (m, 2H), 1.05 (t,J=7.2 Hz, 8H, Et₃NH⁺).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(69)

To a suspension of 68 (2.55 g g, 2.67 mmol) in 25 mL of anhydrous DMFwas added triethylamine (1 mL) followed by PFP-TFA in several portionsover the course of 4 hours until no more starting material was found byHPLC analysis. A total of 0.91 mL (5.3 mmol) of PFP-TFA was added. Theprecipitate was collected by filtration, washed with acetone (3×20 mL)and dried to afford 2.40 g (95%) of PFP ester 69 as a red-purple solid.¹H NMR (DMSO-d6) δ 12.54 (s, 1H), 11.80 (s, 1H), 8.5-8.2 (m, 3H), 8.07(d, J=8.7 Hz, 1H), 7.89 (d, J=9 Hz, 2H), 7.74 (br d, J=8.1 Hz, 1H), 7.59(s, 1H), 7.43 (d, J=8.7 Hz, 1H), 7.37 (br s, 1H), 7.11 (s, 1H), 6.98 (d,J=9.3 Hz, 2H), 4.68 (t, J=7.5 Hz, 2H), 4.22 (t, J=6.3 Hz, 2H), 4.07 (t,J=6.3 Hz, 2H), 3.66 (t, J=6.2 Hz, 2H), 3.48 (t, J=7.2 Hz, 2H), 3.31 (t,J=7.5 Hz, 2H), 3.16 (s, 3H), 2.04 (s, 3H), 1.93 (m, 2H).

N-(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhex-1-yl)-3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(70)

A solution of O-DMT-, N-Fmoc-protected 6-aminohexan-1-ol (S. Mahajan(2006)) (0.55 g, 0.86 mmol) in a mixture of DMF (6 mL) and triethylamine(6 mL) was heated at 80° C. for 1 h, then concentrated and re-dissolvedin a fresh portion of DMF (30 mL) supplemented with triethylamine (0.45mL). To this solution was added 0.74 g (0.78 mmol) of 69 and thereaction was stirred at 50° C. for 5 h and then at room temperature for18 h. DMF was removed under reduced pressure and the resultant residuetriturated with methanol. Filtration of the obtained solid followed bywashing with methanol and drying yielded 0.885 g (87%) of compound 70 asa purple solid. ¹H NMR (DMSO-d6) δ 11.78 (s, 1H), 11.64 (s, 1H), 8.45(br t, 1H), 8.33 (s, 1H), 8.20 (m, 2H), 8.08 (d, J=8.4 Hz, 1H), 7.90 (d,J=9.3 Hz, 2H), 7.75 (br d, J=6.9 Hz, 1H), 7.4-7.25 (m, 11H), 7.08 (s,2H), 6.98 (d, J=9.3 Hz, 2H), 6.89 (d, J=9 Hz, 4H), 4.65 (t, J=7.5 Hz,2H), 4.22 (t, J=6.3 Hz, 2H), 4.07 (t, J=6 Hz, 2H), 3.73 (s, 6H), 3.66(t, J=6.2 Hz, 2H), 3.43 (t, J=7.2 Hz, 2H), 3.28 (m, 4H), 3.16 (s, 3H),2.96 (t, J=6 Hz, 2H), 2.04 (s, 3H), 1.94 (m, 2H), 1.55 (m, 4H), 1.32 (m,4H).

N-(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhex-1-yl)-3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-hydroxypropyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yI]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(71)

To a solution of 70 (0.61 g, 0.52 mmol) in 20 mL of DMF was added 10 mLof ethanol followed by 5 mL of 1 N NaOH. The reaction was heated at 50°C. with stirring for 30 min, then concentrated and the resultant residueresuspended in methanol (30 mL). The obtained solid was collected byfiltration, washed with methanol (2×30 mL) and dried to afford 0.562 g(95%) of 71 as a purple solid. ¹H NMR (DMSO-d6) δ 11.77 (s, 1H), 11.64(s, 1H), 8.45 (br t, 1H), 8.31 (s, 1H), 8.20 (m, 2H), 8.08 (d, J=8.4 Hz,1H), 7.88 (d, J=9 Hz, 2H), 7.72 (br d, J=6.9 Hz, 1H), 7.4-7.25 (m, 11H),7.07 (s, 2H), 6.97 (d, J=9.3 Hz, 2H), 6.87 (d, J=8.7 Hz, 4H), 4.65 (t,J=7.5 Hz, 2H), 4.21 (t, J=6.3 Hz, 2H), 3.71 (s, 6H), 3.62 (t, J=6.3 Hz,2H), 3.49 (m, 2H), 3.39 (t, J=7.2 Hz, 2H), 3.27 (m, 4H), 3.16 (s, 3H),2.92 (t, J=6 Hz, 2H), 1.76 (m, 2H), 1.54 (m, 4H), 1.31 (m, 4H).

N-(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhex-1-yl)-3,6,7,8-tetrahydro-6-[[3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-{[4-oxo-4-(pentafluorophenoxy)butanoyl]oxy}propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(72)

Compound 71 (0.56 g, 0.49 mmol) was dried by co-evaporation withanhydrous DMF and then re-dissolved in a fresh portion (10 mL) of DMF.Triethylamine (0.3 mL), N-methylimidazole (0.1 mL) and succinicanhydride (0.10 g, 1.0 mmol) were added and the reaction was stirred at50° C. for 7 h until no starting material was found by HPLC analysis.The reaction was cooled and treated with triethylamine (0.2 mL) andPFP-TFA (0.21 mL, 1.22 mmol). After being stirred for 2 h the reactionwas concentrated under reduced pressure and the resultant semi-solidresuspended in cold methanol (50 mL). The insoluble material wascollected by filtration, washed with methanol (3×10 mL) and dried toafford 0.642 g (93%) of PFP ester 72 as a red-purple solid. ¹H NMR(DMSO-d6) δ 11.78 (s, 1H), 11.64 (s, 1H), 8.45 (br t, 1H), 8.32 (s, 1H),8.20 (m, 2H), 8.08 (d, J=8.4 Hz, 1H), 7.86 (d, J=9 Hz, 2H), 7.76 (br d,J=6.9 Hz, 1H), 7.4-7.25 (m, 11H), 7.08 (s, 2H), 6.97 (d, J=9.3 Hz, 2H),6.88 (d, J=8.4 Hz, 4H), 4.65 (t, J=7.5 Hz, 2H), 4.22 (t, J=6.3 Hz, 2H),4.14 (t, J=5.4 Hz, 2H), 3.72 (s, 6H), 3.67 (m, 2H), 3.40 (m, 2H), 3.28(m, 4H), 3.15 (s, 3H), 3.07 (t, J=6 Hz, 2H), 2.96 (t, J=6 Hz, 2H), 2.79(t, J=6 Hz, 2H), 1.95 (m, 2H), 1.54 (m, 4H), 1.33 (m, 4H).

Preparation of Polystyrene Support ID #480

Aminomethyl-polystyrene support (33 umol/g amine capacity, AppliedBiosystems, PN 360865C) (5.0 g) was added to a solution of 72 (0.12 g,0.14 mmol, 17 umol/g offering) and triethylamine (0.5 mL) in 25 ml ofDMF. The suspension was swirled on an orbital shaker for 22 h thenfiltered and washed with DMF. To block unreacted aminogroups the supportwas suspended in pyridine (22.5 mL), treated with acetic anhydride (2.5mL) and swirled for 30 min. The blocking reagents were removed byfiltration and the support was washed with several 50 mL portions of DMFand acetone followed by drying under reduced pressure to affordpolystyrene support ID #480 as a light purple solid. DMT loading was16.1 μmol/g according to a colorimetric acid cleavage test.

Example 6. Preparation of a DSQ-PhosphoramiditeN-ethoxycarbonyl-N-(6-(bis-(4-methoxyphenyl))(phenyl)methoxyhex-1-yl)-3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6-[[3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(73)

To a suspension of compound 70 (0.88 g, 0.74 mmol) in a solution oftriethylamine (1 mL) and N,N-dimethylaminopyridine (0.054 g) in 13 mL ofanhydrous DMF was added with stirring at 50° C. 1 mL ofdiethylpyrocarbonate. Five more portions of diethylpyrocarbonatetotaling 6 mL were added over the course of 6 h. The reaction wasallowed to proceed at room temperature for 2 more days then concentratedand partitioned between CH₂Cl₂ and water. The organic phase was driedover Na₂SO₄ and concentrated. The obtained residue was chromatographedon silica eluting with acetone in ethyl acetate (0-5%). Concentration ofthe main product fractions afforded 0.7 g of a purple amorphous solid.¹H NMR analysis indicated the presence of two compounds, one likely tobe the desired triethoxycabonyl-protected compound 73 (approx. 66%) andthe other one being the dioxoimidazo cyclization product (approx. 33%)as shown in FIG. 9. This mixture was taken to the next step withoutadditional purification.

N-ethoxycarbonyl-N-(6-hydroxyhex-1-yl)-3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6-[[3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(74)

Methanol (3 mL) and water (0.06 mL) were added to a solution of 73 (0.7g, 0.52 mmol) in CH₂Cl₂ (12 mL) followed by trifluoroacetic acid (0.12mL). After being kept at room temperature for 10 min the reaction wasquenched by adding 0.24 mL of triethylamine and then concentrated. Theobtained residue was chromatographed on silica eluting with 3:4acetone-ethyl acetate. Concentration of the main product fractionsafforded 0.52 g of a dark purple amorphous solid. ¹H NMR spectrum wasconsistent with the product being a mixture of the desired 74 and thedioxoimidazo analog (FIG. 9). This mixture was taken to the next stepwithout additional purification.

N-ethoxycarbonyl-N-(6-{(cyanomethoxy)[di(propan-2-yl)amino]phosphanyl}oxyhex-1-yl)-3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6-[[3-(ethoxycarbonyl)-3,6,7,8-tetrahydro-6[(2-[(E)-(4-{[3-(acetyloxy)propyl](methyl)amino}phenyl)diazenyl]-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-2-yl]carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxamide(75)

To a solution of 74 (0.51 g, 0.5 mmol) in 10 mL of anhydrous CH₂Cl₂ wasadded diisopropylammonium tetrazolide (0.077 g) followed by 0.21 g (0.66mmol) of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite. Thereaction was stirred for 3 h then diluted with CH₂Cl₂ (100 mL) andextracted with 50 mL of saturated NaHCO₃. The organic phase was washedwith brine and dried over MgSO₄. Concentration of the extract gave anamorphous solid foam which was then re-dissolved in small amount(approx. 6 mL) ethyl acetate followed by diluting with hexane (100 mL).The resultant precipitate was collected by filtration, washed withhexane and dried to afford 0.56 g of phosphoramidite 75 as a mixturewith the dioxoimidazo analog shown in FIG. 9. ³¹P NMR (DMSO-d6) δ145.90, 145.87.

Example 7. Preparation of Oligonucleotide Conjugates by Post-SyntheticModification of Amine-Tailed Oligonucleotides2-{(E)-[4-Phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazole-6-carboxylicAcid (76)

40% Nitrosylsulfuric acid (0.8 mL) was added over the course of 1 min toa stirred suspension of 2-amino-1,3-benzothiazole-6-carboxylic acid(0.625 g, 3.22 mmol) in a mixture of acetic (8 mL) and propionic (8 mL)acids at approx. 5° C. The reaction was then stirred at room temperaturefor 2 h before being used in the next step. A solution of sulfamic acid(0.1 g) in water (30 mL) was combined with a solution of 52 (0.89 g,3.87 mmol) in methanol (30 mL). The resultant emulsion was cooled toapprox. 5° C. and then slowly (approx. 2 min) added to the diazoniumsalt from the first step. The obtained purple mixture was stirred at0-4° C. for 2 h, at room temperature for 1 h, then diluted with water(300 mL) and filtered to collect the precipitated product. Theprecipitate was washed with water and dried to afford 0.69 g (49%) ofcrude dye 76 as a black solid. ¹H NMR (DMSO-d6) δ 12.25 (br s, 1H), 8.54(s, 1H), 8.32 (m, 2H), 7.98 (skewed d, J=8.7 Hz, 1H), 7.87 (skewed d,J=8.7 Hz, 1H), 7.63 (m, 3H), 3.95 (m, 2H), 3.63 (m, 2H), 2.09 (m, 4H).

Pentafluorophenyl2-{(E)-[4-Phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazole-6-carboxylate(77)

Acid 76 (0.69 g, 1.58 mmol) was first dried by co-evaporation with DMF(20 mL) supplemented with triethylamine (0.5 mL) then re-dissolved in amixture of CH₂Cl₂ (20 mL) and triethylamine (0.5 mL). PFP-TFA (0.4 mL,2.33 mmol) was added in one portion. The reaction was stirred for 1 hthen concentrated and the obtained solid re-suspended in cold methanol(20 mL). The resultant precipitate was collected by filtration, washedwith methanol (2×5 mL) and dried under reduced pressure to afford 0.76 g(80%) of PFP ester 77 as a black solid. ¹H NMR (CDCl₃) δ 8.65 (s, 1H),8.40 (m, 2H), 8.21 (skewed d, J=8.4 Hz, 1H), 8.03 (skewed d, J=8.4 Hz,1H), 7.55 (m, 3H), 4.00 (m, 2H), 3.57 (m, 2H), 2.18 (m, 4H).

3,6,7,8-Tetrahydro-6-[(2-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylicAcid (78)

To a solution of3,6,7,8-tetrahydro-benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylic acid (0.25mmol) in 2 mL of DMF was added 0.5 mL of triethylamine followed by 0.15g (0.25 mmol) of PFP ester 77. The mixture was stirred for a few minutesto give an almost complete solution before product precipitation began.The suspension was stirred at room temperature for 3 h, thenconcentrated on a rotary evaporator and the resultant black residue wasre-suspended in methanol (7 mL). The obtained precipitate was collectedby filtration, washed with methanol (2×2 mL) and dried under reducedpressure to afford 0.158 g (100%) of compound 78 (partialtriethylammonium salt) as a black solid. ¹H NMR (DMSO-d6) δ 11.69 (br s,1H), 8.32 (m, 2H), 8.23 (s, 1H), 7.92 (d, J=8.4 Hz, 1H), 7.61 (m, 4H),7.27 (br s, 1H), 6.93 (s, 1H), 4.17 (t, J=7.8 Hz, 2H), 3.92 (m, 2H),3.61 (m, 2H), 3.25 (t, J=8.1 Hz, 2H), 2.76 (m, 2.5H, Et₃NH⁺), 2.08 (m,4H), 1.06 (t, J=7.2 Hz, 4.1H, Et₃NH⁺).

Pentafluorophenyl3,6,7,8-tetrahydro-6-[(2-{(E)-[4-phenyl-2-(pyrrolidin-1-yl)-1,3-thiazol-5-yl]diazenyl}-1,3-benzothiazol-6-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrole-2-carboxylate(79)

PFP-TFA was added in two, 0.05 mL portions over the period of 2 h to asolution of 78 (0.154 g, 0.25 mmol) and triethylamine (0.1 mL) in 5 mLof anhydrous DMF. The initial solution turned into a suspension by theend of this time. The suspension was cooled on ice, the insolublematerial was cooled by filtration, washed with methanol and dried toafford 0.15 (76%) of PFP ester 79 as a black metallic solid. ¹H NMR(DMSO-d6) δ 12.52 (s, 1H), 8.32 (m, 2H), 8.26 (s, 1H), 7.94 (d, J=8.1Hz, 1H), 7.68 (d, J=8.1 Hz, 1H), 7.61 (m, 3H), 7.55 (s, 1H), 7.38 (br s,1H), 4.22 (t, J=7.8 Hz, 2H), 3.93 (m, 2H), 3.62 (m, 2H), 3.25 (tobscured by water signal, J=8.1 Hz, 2H), 2.08 (m, 4H).

Post-Synthetic Conjugation of DSQ Groups by Reaction of the6-amino-2-hydroxyhexyl-modified Octadeoxythymidylate with PFP Esters 44,77, 79 and Dabcyl

To a solution of 3′-(6-amino-2-hydroxyhexyl)-octadeoxythymidylate(approx. 60 nmol), which had been prepared by oligonucleotide synthesisstarting from the PS support ID #394 (Example 2) followed by C18 HPLCpurification using triethylammonium bicarbonate buffer, in 20 μL of DMSOwas added 0.7 mg (1.1 μmol) of PFP ester 44, 77 or 79 and 0.5 μL oftriethylamine. After being kept at room temperature (55° C. in case ofPFP ester 79) for 20 h the reaction was diluted with 1×HPLC buffer,centrifuged to spin down unreacted PFP ester and chromatographed on aC18 HPLC column eluting with a gradient of CH₃CN in 0.1 Mtriethylammonium bicarbonate buffer (pH ^(˜)9). The product peak wascollected and dried in a SpeedVac vacuum concentrator under reducedpressure. Dabcyl conjugation using Quencher-470 PFP ester (ELITechGroupInc., product # M830572) was performed analogously. The identities andpurity of the products were confirmed by ESI mass-spectrometry.

Example 8. Absorption Spectra of Exemplary OctadeoxythymidylateConjugates of the Disclosure

Absorption spectra of various compounds in accordance with preferredembodiments of the present disclosure conjugated tooctadeoxythymidylate, as shown in FIG. 11, were obtained using theinstrumentation and measurement conditions described in Table 4 below.

TABLE 4 Instrumentation and measurement conditions Instrument PE Lambda2S UV-VIS spectrophotometer Conc. (mM) 0.01-0.03 Diluent 50 mM Tris-HClpH 8.5 Temperature (° C.) Ambient

FIG. 11A-11C show UV-VIS absorption spectra of representativeoligonucleotide conjugates in accordance with preferred embodiments.

Example 9. Oligonucleotide Synthesis

All oligonucleotides and oligonucleotide conjugates used in Examples10-14 were synthesized using the instrumentation, synthesis andpurification conditions described in Table 5 below.

TABLE 5 Instrumentation and synthesis conditions Instrument ABI 3900 DNAsynthesizer Scale 200 nmol Deblocking 3% TCA Amidite conc. 100 mM(double coupling for all dye and quencher amidites) Activator5-Ethylthiotetrazole Oxidation I₂/Pyridine/Water CappingAc₂O/Pyridine/Melm Deprotection conc. NH₄OH, 25% EtOH (70° C., 2 h)Purification 4.6 × 250 mm Luna C18 RP HPLC (2 mL/min, gradient of CH₃CN,triethylammonium bicarbonate buffer, pH ~9) Mass Thermo Scientific, LCQFleet, ESI spectroscopy (TEA-HFIPA buffer)

The mass spectrometry for the d(T₈)-3′-DSQ conjugates is listed in Table6 below.

TABLE 6 Summary of ESI mass- spectroscopy of the d(T₈)-3′-DSQ conjugatesConjugate DSQ MW MW Purity Name ID # Calc. (Da) Observed (Da) (%) T8-473473 3225.5 3225.3 95.97 T8-475 475 3311.5 3311.5 97.47 T8-476 476 3329.63329.4 100 T8-477 477 3243.5 3243.2 100 T8-478 478 3299.6 3299.3 100T8-479 479 3306.9 3306.6 98.23 T8-480 480 3271.6 3271.3 100 T8-481 4813350.6 3350.8 95.67 T8-482 482 2977.2 2977.0 100 T8-483 483 3275.53275.4 100 T8-484 484 3074.3 3074.0 100 T8-485 485 3257.5 3258.4 95.09T8-486 486 3295.57 3295.6 98.92 T8-487 487 3092.35 3092.0 100 T8-488 4882908.16 2907.8 99.02 T8-489 489 2890.12 2896.9 100 T8-490 490 3150.413150.3 98.57 T8-77  77 2984.25 2983.7 100 T8-79  79 3168.45 3168.2 98.50T8-44  44 2927.18 2927.2 96.79 T8-Dabcyl Dabcyl 2818.03 2817.9 100

Example 10. Thermal Denaturation of DSQ-Labeled TaqMan Probes

To evaluate duplex-stabilizing and fluorescence quenching properties ofnew DSQ derivatives, two TaqMan probes labeled with FAM or AP525 wereinvestigated. The probes (200 nM) FAM-5′-G*CAGGTTCCGGTTTTG-DSQ (SEQ IDNO:1) or AP525-5′-G*ACCACGTACCGCATTG-DSQ (SEQ ID NO:2) probes werehybridized in a PCR buffer with 1 μM of the respective complement (FAMcomplement: 5′-TCAAAACCGGAACCTGCT (SEQ ID NO:3); AP525 complement:TCAATGCGGTACGTGGTCT (SEQ ID NO:4) by briefly heating the solution to 80°C., cooling to 20° C., then ramping to 90° C. while monitoringfluorescence. Background fluorescence was measured at 90° C.,temperature at which all duplexes have been completely denatured.Instrumentation and measurement conditions were as shown in Table 7below. Results are summarized in FIGS. 12 and 13.

TABLE 7 Instrumentation and experimental conditions Instrumentation andmeasurement conditions Instrument Varian Cary Eclipse Fluorimeter Datamode Fluorescence Scan mode Emission X Mode Wavelength (nm) Ex.Wavelength (nm) 496 (FAM), 527 (AP525) Em. Wavelength (nm) 517 (FAM),549 (AP525) Ex. Slit (nm) 5 Em. Slit (nm) 5 Measurement 20.0-90.0, 1°C./min ramp temperature (° C.) 1X PCR buffer 40 mM NaCl, 10 mM Tris, 5mM MgCl₂, pH 8.9

FIG. 12 shows a melting temperature comparison for duplexes formedbetween two DNA probes and their respective complementary targets. Theprobes are labeled with various exemplary DSQ derivatives at the 3′ endand one of the fluorophores, FAM or AP 525, at the 5′ end. For referencepurposes, probes (ID #385), which have the traditional structure of boththe MGB and the quencher (CDPI₃+Eclipse quencher, FIG. 18), are alsoincluded in the comparison. It is clear that probes with certain DSQderivatives (e.g. modification ID #473, 477, 480, 485) have meltingtemperatures that are equal or exceed the values of the referenceprobes. This confirms that the truncated MGB (CDPI₂ in this case) canregain its full duplex-stabilizing ability when extended with certaintypes of diaryl-azo derivatives of Formula I. Notably, the probeslabeled with DSQ ID #485 outperform the reference probes (ID #385)indicating that, at least in this particular sequence context, thediaryl-azo groups bring more to duplex stability than the displaced CDPIunit. The least efficient duplex-stabilizing DSQ derivatives (ID #479,481, 483) contained either a 1,4-substituted phenyl or an2,4-substituted-N-methylpyrrol ring as the Ar¹ group of formula III. Thelatter result is unexpected in view of the report by Ong et al. (2012)on the properties4-(1-methylpyrrol-2-yl)azo-1-methylpyrrole-2-carboxylates.

FIG. 13 addresses the fluorescence quenching properties of preferredembodiments of the DSQ derivatives. In this test, backgroundfluorescence of unhybridized probes is the measure of quenchingefficiency. As expected, the derivatives with the best spectral overlapbetween fluorophore's emission (FAM emission maximum—515 nm, AP525emission maximum—549 nm) and quencher's absorption (FIG. 11) demonstratethe lowest background fluorescence thus confirming that the mainquenching occurs via the FRET mechanism (Didenko (2001)). Some DSQanalogs (e.g. SDQ ID #476, 477, 478 and 480) have the backgroundfluorescence as low or lower as the reference Eclipse quencher (part ofmodification ID #385). These results confirm that the general quenchingproperties of aryl-azo compounds are not affected by the incorporationinto the DSQ structure according to the statements of thisspecification.

Example 11. Fluorescence Melting Analysis of d(T₈)-DSQ/FAM-d(A₈CC)Duplexes

To evaluate duplex stabilizing effect of representative DSQ derivativesin an A/T-rich sequence context, a short d(T₈)/d(A₈CC) duplex wasinvestigated by fluorescence melting analysis. 3′-DSQ-TTTTTTTT(d(T₈)-DSQ) conjugates were combined with complement (5′-FAM-AAAAAAAACC(SEQ ID NO:5)) in the buffer specified in Table 8 and the solutionequilibrated to 10° C., and dissociation curves acquired by monitoringfluorescence from 10-70° C. at a ramp of 1° C./min. Duplex denaturationwas monitored by detecting fluorescence change over a thermal ramp.Melting temperature (Tm) was calculated by finding second derivativemaximum of the raw melt curve. Instrumentation and measurementconditions were as shown in Table 8 below. Results are summarized inTable 9 and FIG. 14.

TABLE 8 Instrumentation and experimental conditions Instrumentation andmeasurement conditions Instrument Varian Cary Eclipse Fluorimeter Datamode Fluorescence Ex. Wavelength (nm) 496 (FAM), 527 (AP525) Em.Wavelength (nm) 517 (FAM), 549 (AP525) Ex. Slit (nm) 5 Em. Slit (nm) 5Measurement 10-70° C., 1° C./min temperature (° C.) Buffer 100 mM NaCl,10 mM MgCl₂, 10 mM Na2-PIPES, pH 7.0 T8 conjugate Concentration 0.2 μMComplement Concentration 0.1 μM

TABLE 9 Tm data Sequence ID Δ Tm General DSQ Structure: DSQ Tm relativeto (CDPI)_(n)—(O═)C—Ar¹—N═N—Ar² Name ID # (° C.) T8-Dabcyl n —(O═)C—Ar¹——Ar² T8-473 473 45.6 30.0 2 Indolyl (Dimethylamino)phenyl T8-475 47544.3 28.7 2 Indolyl (Pyrrolidino,dimethoxy)phenyl T8-476 476 21.9 6.3 2Benzothiazolyl (Pyrrolidino,dimethoxy)phenyl T8-477 477 45.5 29.9 2Benzothiazolyl (Dimethylamino)phenyl T8-478 478 44.2 28.6 2Benzothiazolyl (Dimethylamino)phenyl T8-479 479 34.7 19.1 2 Phenyl(Pyrrolidino,dimethoxy)phenyl T8-480 480 45.3 29.7 2 Benzothiazolyl(Dimethylamino)phenyl T8-481 481 26.7 11.1 2 Phenyl(Dimethylamino)phenyl-azo-phenyl T8-482 482 26.7 11.1 2 N/A N/A T8-483483 35.6 20.0 2 pyrrolyl (Pyrrolidino,dimethoxy)phenyl T8-484 484 28.813.2 1 Indolyl Pyrrolidinothiazolyl T8-485 485 44.8 29.2 2 IndolylPyrrolidinothiazolyl T8-486 486 47.6 32.0 2 Benzothiazolyl JulolidinylT8-487 487 16.6 1.0 1 Benzothiazolyl Pyrrolidinothiazolyl T8-488 48819.3 3.7 0 Benzothiazolyl Pyrrolidinothiazolyl T8-489 489 18.4 2.8 0Indolyl Pyrrolidinothiazolyl T8-490 490 23.2 7.6 1 Indolyl(Pyrrolidino)phenylthiazolyl T8-77 77 23.9 8.3 0 Benzothiazolyl(Pyrrolidino)phenylthiazolyl T8-79 79 20.7 5.1 1 Benzothiazolyl(Pyrrolidino)phenylthiazolyl T8-44 44 16.5 0.9 0 BenzothiazolylJulolidinyl T8-dabcyl Dabcyl 15.6 0.0 0 Phenyl (Dimethylamino)phenyl

FIG. 14A shows the effects of various DSQ ligands on the d(T₈)/d(A₈)duplex stability and FIG. 14B shows the effects of various DSQ ligandson the d(T₈)/d(A₈) duplex melting temperature, another demonstration ofduplex-stabilizing properties of new DSQ derivatives. In this example, ashort d(T₈)/d(A₈CC) duplex is used to enhance the differences betweenthe derivatives. As a control, a CDPI₂-labeled d(T₈) oligonucleotide isalso included. The d(A₈CC) complement is labeled with a fluoresceingroup at the 5′-end, which is quenched in the duplex form and unquenchedupon duplex denaturation allowing for fluorescence-based meltingdetection. It can be seen that the DSQ analogs, composed of certainaryl-azo moieties and two CDPI units (e.g. DSQ ID #473, 475, 477, 478,479, 480, 483, 485 and 486), outperform the control CDPI₂-conjugate thusconfirming that the aryl-azo moiety effectively contributes to theduplex stabilization according to the statements of this specification.In one example, the DSQ ID #484 conjugate, which has a diaryl-azo moietycoupled to a single CDPI unit, outperforms the control CDPI₂-conjugatesshowing that this particular structure has especially high affinity forDNA duplex.

Example 12. Evaluation of Sequence Effect on Duplex Stabilization by DSQID #477

To establish the stabilizing contribution of the DSQ (ID #477) moiety tothe duplex, sequences (Table 11) with varying G/C content in theinferred binding region of the DSQ moiety were evaluated. Forcomparison, CDPI₂ (ID #482), CDPI₃ (ID #391), MGB-Quencher (ID#385)-labeled and unmodified oligonucleotides were also tested. Duplexeswere heated briefly to 80° C., equilibrated at 20° C., and then rampedto 70 or 90° C. while monitoring UV absorbance. Instrumentation andmeasurement conditions were as shown in Table 10 below. Results aresummarized in Table 11 and FIG. 15.

TABLE 10 Instrumentation and experimental conditions Instrumentation andmeasurement conditions Instrument Cary Bio 400 Wavelength monitor 260 nmMeasurement 20-70 or 20.0-90.0, temperature (° C.) 1° C./min ramp Buffer1 20 mM Na₂-PIPES, pH 8 Buffer 2 10 mM Na₂-PIPES, 100 mM NaCl, 10 mMMgCl₂, pH 7 Test Oligo Concentration 3 μM Complement Concentration 6 μM

TABLE 11 SEQ Tm (° C.) ID Sequence 5′- 5′- 5′- 5′- NO: (5′-3′) none[482] [391] [385] [477]  6 ACACAAGCTACA 56.85 60.38 73.05 65.56 71.00  7TTATATGCCACG 53.50 61.37 73.89 68.87 63.61  8 TACACTGGACAT 55.75 61.4466.18 60.40 66.80  9 CAGAGCTTACAT 53.75 58.77 70.51 64.68 53.78 10GCTCTGTTAAGT 50.27 52.19 57.69 56.53 54.20 11 GAAAACACCGTC 56.96 62.7573.99 69.36 69.91 12 TCCTGAGTCAAC 55.29 60.56 61.66 61.15 53.42 13CGCTAAATCCTG 55.91 61.83 75.16 67.17 71.78 14 TGTTCTACCGAG 54.95 62.5273.78 68.68 68.94 15 CGAAATACCCTG 53.93 63.58 76.78 70.64 72.02

In order to demonstrate that new DSQ moieties can stabilize DNA duplexeswith different sequence context, the test shown in FIG. 15 and Table 10was performed. A set of DSQ (ID #477)-oligonucleotide conjugates withdifferent G/C content in the putative DSQ-binding region was prepared.For comparison, CDPI₂ (ID #482), CDPI₃ (ID #391), MGB-Quencher (ID#385)-labeled and unmodified oligonucleotides were also tested. In allbut two cases, the DSQ ligand outperformed the control CDPI₂ moiety byup to 10° C. thus confirming the earlier results with the T8 conjugates(Example 11). The two sequences (SEQ ID NO:9 and SEQ ID NO:12) withlower than the control CDPI₂ melting temperatures had a significantinternal secondary structure, over-stabilized by the presence of the DSQmoiety, as shown in FIG. 15 This type of MGB-stabilized secondarystructure is expected and should be avoided by thorough sequenceanalysis. Aside the two outliers, the obtained results confirm that thenew DSQ derivatives are suitable for DNA duplex stabilization of varioussequences and G/C content.

Example 13. Real-Time PCR Using DSQ-Labeled TaqMan Probes

To evaluate PCR performance of new DSQ derivatives, PCR was conductedusing two DSQ-labeled TaqMan probes (TaqMan assay). PCR Monoreagentformulation and PCR conditions are shown in Table 12. Result aresummarized in Table 13 and FIG. 16.

TABLE 12 PCR formulation and PCR conditions Instrument ELITe InGeniusKPC primers AATAAATCATAAGCAGACTGGGCAGTCGG (SEQ ID NO: 18)AATAAATCATGTCATTTGCCGTGCCATAC (SEQ ID NO: 19) IC2 primersCTCATTTTTTCTACCGGAGATCTTGT (SEQ ID NO: 20) CTGCACGGACCAGTTACTTTACG(SEQ ID NO: 21) KPC probe FAM-G*CAGGTTCCGGTTTTG-MGBQ (SEQ ID NO: 22)IC2 probe AP525-G*ACCACGTACCGCATTG-MGBQ (SEQ ID NO: 23) Primer   500 nMconcentration  (nM) Probe    100 nM concentration (nM) Magnesium    3concentration (mM) Enzyme     3 U/reaction concentration ThermocyclingPrecycle: 95° C., 240 sec   40 cycles of PCR: Denature: 95° C., 10 secAnneal: 56° C., 30 sec Extend: 73° C., 15 sec KPC target 1000 copies/PCR reaction concentration IC2 target 1000 copies/PCR reaction concentration

TABLE 13 Summary of PCR data Ct Signal at Signal/ DSQ KPC* IC* cycle #45Background Background ID # (Fluorophore) (AP525) KPC* IC* KPC* IC* KPC*IC* 385 28.6 (FAM) 30.3 6306 4551 1237 543 5.1 8.4 473 28.4 (FAM) 29.77469 9960 2329 3829 3.2 2.6 475 28.5 (FAM) 29.8 6914 7653 1564 1719 4.44.5 476 29.2 (FAM) 30.1 5525 5650 1038 479 5.3 11.8 477 28.7 (FAM) 30.45772 6289 909 438 6.4 14.4 477 29.7 (AP593) 27.9 3335 6994 329 724 10.19.7 477 28.3 (AP639) 27.7 18984 7360 1665 828 11.4 8.9 478 29.2 (FAM)30.8 6125 5169 1030 447 5.9 11.6 479 29.3 (FAM) 30.8 5145 5252 1014 8575.1 6.1 480 28.9 (FAM) 30.8 5428 5119 832 374 6.5 13.7 481 29.8 (FAM)31.4 4526 4969 993 773 4.6 6.4 *All values are averages of three or morereplicates

FIG. 16 shows PCR performance of representative conjugates of thedisclosure. Two TaqMan probes, one (KPC) labeled with FAM and the otherone (IC2) with AP525 at the 5′-end (FIG. 12), were synthesized using theDNA synthesis supports described in this specification. As a control,the same two probes were also synthesized using the traditionalMGB-Quencher reagent (modification ID #385). Target DNAs amplificationwas performed in the presence of the probes, target-specific primers andTaq polymerase. To evaluate the performance, threshold cycle (Ct),baseline fluorescence (background), fluorescence signal (fluorescence atcycle 45) and signal-to-background ratio were compared as summarized inTable 12 and FIG. 16. Based on the Ct values, the new probes providedsimilar amplification efficiency and overall test sensitivity as thecontrol probe with Ct values generally varying within one amplificationcycle. Background fluorescence, with the exception of the DSQ ID 473 and475 were same as or lower than the background of the control probe. As aconsequence of the low background, some of the new DSQ probesdemonstrated improved Signal-to-Background ratios (e.g DSQ ID #476, 477,478 and 480). These results demonstrate that the new DSQ derivatives aresuitable for PCR applications with their performance comparable orbetter than the traditional technology.

Simultaneous amplification and detection of multiple targets is anessential requirement for multiplex PCR applications. This can only beenabled if multiple fluorophores with wide range of emission spectra arecombined in one PCR reaction. In order to demonstrate that new DSQderivatives are also suitable for PCR applications with red-shiftedfluorophores an experiment was performed in which, the KPC probe waslabeled either with AP593 (emission maximum 613 nm) or AP639 (emissionmaximum 655 nm) dye at the 5′-end and the DSQ ID #477 at the 3′-end. Theobtained results (summarized in Table 12) confirmed that the KPC targetwas successfully amplified and efficiently detected with Ct values,background fluorescence and signal-to-background ratios similar orbetter to those of the FAM-labeled probe.

Example 14. Evaluation of Hybridization-Triggered Fluorescence ofDSQ-Labeled Probes

To evaluate fluorogenic properties of oligonucleotides of Formula VII,the duplex fluorescence at 50° C. (Signal), single strand fluorescenceat 50° C. (Background) and Signal-to-background ratio (S/B) of twooligonucleotides labeled at the 5′-end with DSQ ID #473 and eitherFAM-HEG (ELITechGroup Inc., product #M830100) or AP525-HEG (ELITechGroupInc., product # M100104) were synthesized and tested. To generate afluorescent signal the DSQ-FAM (or AP525)-5′-TAAAAGGTGTAC (SEQ ID NO:24)(200 nM) were hybridized in a PCR buffer with 1 lIM of the complement(TTGTACACCTTTTATT (SEQ ID NO:25)) by briefly heating the solution to 80°C., cooling to 20° C., then ramping to 90° C. while monitoringfluorescence. Background was measured in the absence of complement.Instrumentation and measurement conditions were as shown in Table 14below. Results are summarized in Table 15 and FIG. 17A-17B.

TABLE 14 Instrumentation and measurement conditions Instrument VarianCary Eclipse Fluorimeter Data mode Fluorescence Scan mode Emission XMode Wavelength (nm) Ex. Wavelength (nm) 496 (FAM), 527 (AP525) Em.Wavelength (nm) 517 (FAM), 549 (AP525) Ex. Slit (nm) 5 Em. Slit (nm) 5Measurement 20.0-90.0, 1° C./min ramp temperature (° C.) 1X PCR buffer40 mM NaCl, 10 mM Tris, 5 mM MgCl₂, pH 8.9

TABLE 15 Background Signal S/B FAM 21.5 128.7 6.0 AP525 45.4 629.5 13.9

The new DSQ derivatives can also be used for the preparation ofhybridization probes and primers, which rely on spatial separationbetween the quenching and fluorescent moieties upon hybridization totarget to generate fluorescent signal. In one particular configuration,the DSQ and fluorophore moieties are positioned at the same end of anoligonucleotide. In such configuration, spatial separation between thequencher and fluorophore is achieved via the DSQ binding within theduplex minor groove. Such conjugates can be synthesized starting from aDSQ synthesis support followed by a fluorophore phosphoramidite that issuitable for internal incorporation (for example FAM-HEG and AP525-HEGphosphoramidites available from ELITechGroup Inc.), and then anoligonucleotide sequence. FIGS. 17A and 17B show results of a thermalmelting experiment involving such fluorogenic oligonucleotide labeledwith the DSQ ID #473 in combination with either FAM or AP525fluorophore. Due to its blue-shifted absorption spectrum (FIG. 11), thisDSQ mostly relies on the contact quenching ((U.S. Pat. No. 6,150,097) inits single stranded state. The quenching is eliminated upon DSQ'sconfinement in the minor groove. As a result such oligonucleotidesdemonstrate good fluorescence signal and signal-to-background ratios(Table 14) and are useful as fluorogenic primers or probes depending onwhether the 3′-hydroxyl is present or blocked.

REFERENCES CITED

The following documents and publications are hereby incorporated byreference.

U.S. and Foreign Patent Documents References Cited

The following documents and publications are hereby incorporated byreference.

U.S. and Foreign Patent Documents

-   EP Patent No. 1384789-   US patent application Nos.-   20140335515-   20130030166F-   US patent No.-   RE 38,416-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 5,419,966-   U.S. Pat. No. 5,512,667-   U.S. Pat. No. 5,585,481-   U.S. Pat. No. 5,696,251-   U.S. Pat. No. 5,736,626-   U.S. Pat. No. 5,801,155-   U.S. Pat. No. 5,942,610-   U.S. Pat. No. 6,150,097-   U.S. Pat. No. 6,312,894-   U.S. Pat. No. 6,323,337-   U.S. Pat. No. 6,399,392-   U.S. Pat. No. 6,492,346-   U.S. Pat. No. 6,699,661-   U.S. Pat. No. 6,699,975-   U.S. Pat. No. 6,727,356-   U.S. Pat. No. 6,790,945-   U.S. Pat. No. 6,821,727-   U.S. Pat. No. 6,972,339-   U.S. Pat. No. 7,019,129-   U.S. Pat. No. 7,166,715-   U.S. Pat. No. 7,205,105-   U.S. Pat. No. 7,262,007-   U.S. Pat. No. 7,381,818-   U.S. Pat. No. 7,439,341-   U.S. Pat. No. 7,564,567-   U.S. Pat. No. 7,582,739-   U.S. Pat. No. 7,767,834-   U.S. Pat. No. 7,759,470-   U.S. Pat. No. 7,803,536-   U.S. Pat. No. 7,879,986-   U.S. Pat. No. 7,790,385-   U.S. Pat. No. 8,163,910-   U.S. Pat. No. 8,586,759-   U.S. Pat. No. 8,637,658-   U.S. Pat. No. 9,056,887

Non-Patent References

-   Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley    &Sons (1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996)-   Berge, S. M., et al., Journal of Pharmaceutical Science, 66: 1-19    (1977).-   Boga, C. et al., Org. Biomol. Chem. 14: 7061-7068 (2016)-   Bonnet et al., Proc. Natl. Acd. Sci. USA, 96: 6171-6176 (1999)-   Crisalli and Kool, Bioconjug Chem., 22: 2345-54 (2011)-   Demidov and Frank-Kamenetskii TRENDS in Biochemical Sciences, 29:    62-71 (2004)-   Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL    APPROACH, IRL Press (1991).-   Edmonds et al. in Modern Carbonyl Olefination. Ed. T. Takeda,    Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2004-   Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL    Press (1984)-   T. W. Greene and P. G. Wuts, Greene's Protective Groups in Organic    Chemistry, Wiley, 4nd ed. 2007-   Harrison and Harrison et al., Compendium of Synthetic Organic    Methods, Vols. 1-8, John Wiley and Sons. 1971-1996-   Heid et al., Genome Res. 6: 986-994 (2009)-   Hermanson, Bioconjugate Techniques, Elsevier, 1996, pages 139-140    and 274-275,-   Hymas W C, Hillyard D R J Virol Methods. 156:124-8 (2009)-   Johansson et al. J. Am. Chem. Soc., 124: 6950-6956 (2002)-   Knemeyer and Marme, DNA & Gene Sequences. 1, No. 2: 145-157 (2007)-   Kutyavin et al. Nucl. Acids Res. 28: 655-661(2000)-   Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,    Springer 2007, pages 368-394-   Lukhtanov et al. Bioconjugate Chemistry, 6: 418-426 (1995)-   Lukhtanov et al., Bioconjug Chemistry, 7:564-567 (1996)-   Lukhtanov et al., Nucl. Acids Res., 35: e30 (2007)-   Mahajan, S. et al. Analytical Biochemistry, 351(2), 273-281 (2006)-   Malicka, J. M. et al. Chemistry—A European Journal, 19: 12991-13001    (2013)-   March J. in Advanced Organic Chemistry, Chapter 4”, 4th edition John    Wiley and Sons, New York, 1992 pages 71-124-   Maniatis, Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORY    MANUAL, Cold Spring Harbor Laboratory Press (1982)-   Matayoshi et al., Science, 247:954-958 (1990)-   Morrison et al. Anal. Biochem., 183: 231-244 (1998)-   Ong et al. Org. Biomol. Chem., 10: 1040-1046 (2012)-   Paris et al. Nucleic Acids Res., 38(7): e95 (2010)-   Reddy, et al. (1999), Pharmacol. Therap., 84:1-111 (1999).-   Sambrook, Fritsch & Maniatis, MOLECULAR CLONING: A LABORATORY    MANUAL, Second Edition, Cold Spring Harbor Laboratory Press (1989)-   Sedlak and Jerome, Diagn Microbiol Infect Dis. 75(1):1-4 (2013).-   Smith, March. Advanced Organic Chemistry 6th ed. 2007 by John Wiley    & Sons, Inc. (pages 501-502)-   Tolstrup et al. Nucl. Acids. Res., 31:3758-3762 (2003)-   Tyagi et al. Nat Biotechnol., 14; 303-8 (1996)-   Tyagi et al. Nat Biotechnol., 16: 49-53 (1998)-   Walker et al., Biopolymers, 44: 323-334 (1997)-   Wemmer, D. E., and Dervan P. B., Current Opinion in Structural    Biology, 7:355-361 (1997)-   Zimmer, C & Wahnert, U., Prog. Biophys. Molec. Bio. 47: 31-112    (1986)

What is claimed is:
 1. A quencher reagent for oligonucleotide labelinghaving the formula:

wherein Ar¹ and Ar² are aromatic or hetero-aromatic moieties; Ar³ is anaromatic or hetero-aromatic moiety; R¹ is H, alkyl or alkyl covalentlyconnected to Ar³; X is hydroxyl, a leaving group, a linker with afunctional group or a protected functional group, or a linker connectedto a synthesis solid support; and n is from 0 to
 5. 2. The quencherreagent of claim 1, wherein Ar¹ and Ar² are substituted with a reactivegroup, a linker with a functional group or a protected functional group,or a linker connected to a synthesis solid support.
 3. A method forpreparing an oligonucleotide conjugate labeled with a quencher,comprising: reacting an oligonucleotide with the quencher reagent ofclaim 1 to produce an oligonucleotide conjugate labeled with a quencher.4. The quencher reagent of claim 1 having the formula:


5. A method for preparing an oligonucleotide conjugate labeled with aquencher, comprising: reacting an oligonucleotide with the quencherreagent of claim 4 to produce an oligonucleotide conjugate labeled witha quencher.
 6. The quencher reagent of claim 4, having the formula:

wherein Ar² is an aromatic or hetero-aromatic ring with an exocyclicamino group, and n is between 0 and
 3. 7. The quencher reagent of claim6, wherein the exocyclic amino group is substituted with one or two ofan alkyl, a linker with a functional group or protected functionalgroup, or a linker connected to a synthesis solid support.
 8. A methodfor preparing an oligonucleotide conjugate labeled with a quencher,comprising: reacting an oligonucleotide with the quencher reagent ofclaim 6 to produce an oligonucleotide conjugate labeled with a quencher.9. The quencher reagent of claim 4, having the formula:

wherein X is hydroxyl, —OPFP, or a leaving group, and n is 0, 1 or 2.10. A method for preparing an oligonucleotide conjugate labeled with aquencher, comprising: reacting an oligonucleotide with the quencherreagent of claim 9 to produce an oligonucleotide conjugate labeled witha quencher.
 11. An oligonucleotide conjugate comprising a fluorescencequenching compound and having the formula:

wherein ODN is an oligonucleotide; L is a linking group having from 0 to100 main chain atoms selected from C, N, O, S, P and Si, wherein L caninclude acyclic, cyclic or aromatic groups, or combinations thereof; R¹is H, alkyl, or alkyl covalently connected to Ar³ Ar¹ and Ar² arearomatic or hetero-aromatic moieties; Ar³ is an aromatic moiety; R³ ishydroxyl, a linking group, or a blocking group; and n is from 0 to 5.12. The oligonucleotide conjugate of claim 11, wherein theoligonucleotide further comprises a fluorophore connected to theoligonucleotide.
 13. The oligonucleotide conjugate of claim 12, whereinthe fluorophore is FAM, AP525, AP559, AP593 or AP662.
 14. Theoligonucleotide conjugate of claim 11, wherein the oligonucleotidefurther comprises a minor groove binder connected to theoligonucleotide.
 15. The oligonucleotide conjugate of claim 11, whereinthe oligonucleotide comprises one or more modified nucleobases ormodified bases.
 16. The oligonucleotide conjugate of claim 11, wherein Lis connected to the oligonucleotide at a 3′-end.
 17. The oligonucleotideconjugate of claim 11, wherein L is connected to the oligonucleotide ata 5′-end.
 18. The oligonucleotide conjugate of claim 11, wherein L isconnected to the oligonucleotide at a position other than a 3′- or5′-end.
 19. A method for detecting a target nucleic acid sequence in asample, comprising: contacting the sample with the oligonucleotideconjugate of claim 11, wherein the oligonucleotide conjugate has anucleic acid sequence at least partially complementary to the targetnucleic acid sequence, and wherein the oligonucleotide conjugate furthercomprises a fluorophore; and detecting a fluorescent signal resultingfrom hybridization of the oligonucleotide conjugate to the targetnucleic acid sequence.
 20. The method of claim 19, wherein thefluorescent signal is generated by action of a polymerase having5′-nuclease activity.
 21. The method of claim 19, wherein theoligonucleotide conjugate is a primer or a probe.
 22. The method ofclaim 19, further comprising the step of amplifying the target nucleicacid sequence.
 23. The oligonucleotide conjugate of claim 11 having theformula:

wherein n is 1, 2, or
 3. 24. A method for detecting a target nucleicacid sequence in a sample, comprising: contacting the sample with theoligonucleotide conjugate of claim 23, wherein the oligonucleotideconjugate has a nucleic acid sequence at least partially complementaryto the target nucleic acid sequence, and wherein the oligonucleotideconjugate further comprises a fluorophore; and detecting a fluorescentsignal resulting from hybridization of the oligonucleotide conjugate tothe target nucleic acid sequence.
 25. The method of claim 24, whereinthe fluorescent signal is generated by action of a polymerase having5′-nuclease activity.
 26. The method of claim 24, wherein theoligonucleotide conjugate is a primer or a probe.
 27. The method ofclaim 24, further comprising the step of amplifying the target nucleicacid sequence.
 28. The oligonucleotide conjugate of claim 11 having theformula:

wherein n is 1, 2, or
 3. 29. A method for detecting a target nucleicacid sequence in a sample, comprising: contacting the sample with theoligonucleotide conjugate of claim 28, wherein the oligonucleotideconjugate has a nucleic acid sequence at least partially complementaryto the target nucleic acid sequence, and wherein the oligonucleotideconjugate further comprises a fluorophore; and detecting a fluorescentsignal resulting from hybridization of the oligonucleotide conjugate tothe target nucleic acid sequence.
 30. The method of claim 29, whereinthe fluorescent signal is generated by action of a polymerase having5′-nuclease activity.
 31. The method of claim 29, wherein theoligonucleotide conjugate is a primer or a probe.
 32. The method ofclaim 29, further comprising the step of amplifying the target nucleicacid sequence.